Antibodies that bind to tl1a and their uses

ABSTRACT

The present invention relates to antibodies or fragments thereof that bind to TL1A. More specifically, the present invention relates to an antibody or fragment thereof that binds to TL1A comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 51, and/or a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and/or a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 53; and/or comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 54, and/or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 55 and/or a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 56.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/279,670; filed Sep. 29, 2016, which is a continuation of U.S.application Ser. No. 15/041,953; filed Feb. 11, 2016, which is acontinuation of U.S. application Ser. No. 14/146,566; filed Jan. 2,2014, now U.S. Pat. No. 9,290,576, which claims the benefit of U.S.Provisional Application No. 61/748,201, filed Jan. 2, 2013, which areincorporated by reference herein in their entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:3305_0120004_SL.txt; Size: 101,794 bytes; and Date of Creation: May 4,2017) is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies or fragments thereof thatbind to TL1A. More specifically, the present invention relates to anantibody or fragment thereof that binds to TL1A comprising a heavy chainCDR1 comprising the amino acid sequence of SEQ ID NO: 51, and/or a heavychain CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and/or aheavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 53;and/or comprising a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 54, and/or a light chain CDR2 comprising the amino acidsequence of SEQ ID NO: 55 and/or a light chain CDR3 comprising the aminoacid sequence of SEQ ID NO: 56.

BACKGROUND OF THE INVENTION

TNF-like ligand 1A (TL1A) is a member of the tumor necrosis factor(ligand) superfamily, member 15. TL1A is also known as TNFSF15 and VEGIand was identified in 1999 as an angiogenesis inhibitor that suppressesthe growth of colon carcinomas in vivo (Zhai Y et al., (1999) FASEB J,13(1): 181-9). The protein is abundantly expressed in endothelial cellsand activated cells of the hematopoietic lineage, including monocytes,macrophages, lymphocytes, lamina propria mononuclear cells, dendriticcells and plasma cells but is not expressed in either B or T cells (TanK B et al., (1997) Gene, 204: 35-46; Prehn J L et al., (2007) J Immunol,178: 4033-4038). It is also expressed in kidney, lung, prostate andthymus (Tan K B et al., (1997), supra). It is a ligand for TNFRSF25/DR3and decoy receptor TR6/DcR3 and its expression is inducible by TNF andIL-1α. TNFRSF25/DR3 is a death domain-containing receptor that isupregulated during T cell activation. TL1A induces NF-kappaB activationand apoptosis in TNFRSF25/DR3-expressing cell lines, and in T cells,TL1A can act as a costimulator that increases IL-2 responsiveness andsecretion of proinflammatory cytokines both in vitro and in vivo. Theinteraction of TL1A with DR3 can promote T cell expansion during animmune response (Migone T S et al., (2002) Immunity, 16(3): 479-92). Thesecreted decoy receptor (DcR3), a soluble protein of the tumor necrosisfactor receptor (TNFR) superfamily, blocks the action of TL1A. (Kim S &Zhang L, (2005) J Immunol Methods, 298: 1-8). TL1A has been implicatedas a potential therapeutic target in a number of diseases and disorders.

A major cause of lung inflammation in allergy and asthma is Th2polarization of CD4 T cells with elevated IgE levels and production ofIL-13 by NKT cells. TL1A plays a major role in allergic lunginflammation by co-stimulating IL-4 and IL-13 production in NKT cells.Blocking TL1A and DR3 interaction by TL1A antibody or dominant negativeTL1A mutant abolishes lung inflammation (Fang L et al., (2008) J ExpMed, 205(5): 1037-48). DcR3, the decoy receptor for TL1A is expressed inseveral lung and colon carcinomas and in some normal tissues, thereforesuggesting a role for TL1A in lung and colon carcinomas. In addition,TL1A has also been reported to be angiostatic and to inducemetalloproteinase and IL-8 gene expression (Su W B et al., (2006) ExpCell Res, 312: 266-277; Kang Y J et al., (2005) Cytokine, 29: 229-235).TL1A and DR3 may also be involved in the pathogenesis of atherosclerosisby increasing the production of proinflammatory cytokines and chemokinesand decreasing plaque stability by inducing extracellularmatrix-degrading enzymes (Kang Y J et al., (2005), supra). There is alsoevidence to suggest that TL1A/DR3 is involved in the etiology ofrheumatoid arthritis (Bossen C et al., (2006) J Biol Chem, 281(20):13964-13971).

An association between the expression of TL1A and inflammatory boweldisease has been identified by researchers (Prehn J L et al., (2004)Clin Immunol, 112: 66-77; Bamias G et al., (2003) J Immunol, 171:4868-4874). Crohn's disease, which is a severe inflammatory boweldisorder, is thought to originate from predisposing genetic andenvironmental factors that cause an imbalance of effector(proinflammatory) and regulatory T cell responses, resulting ininflammation of the gastrointestinal mucosa and disease. The TL1A/DR3pathway has been shown to play an important role in intestinal diseases,such as Crohn's disease (Papadakis K A et al., (2005) J. Immunol, 174:4985-4990; Bamias G et al., (2003), supra) and therefore, blockade ofthe TL1A/DR3 pathway may offer therapeutic opportunities in thisdisease.

Death receptors and their ligands play a key role in the maintenance oftissue homeostasis and the physiological regulation of programmed celldeath. Binding of a death ligand induces oligomerization of thereceptor, recruitment of an adapter protein via a conserved cytoplasmicsignalling element termed the death domain, activation of caspases andinduction of apoptosis (Young H A et al., (2006) Proc Natl Acad Sci USA,103(22): 8303-8304). Although death receptors such as Fas/Apo-1/CD95,TNF-R1, TRAIL-R1, TRAIL-R2, or DR3 were initially characterized asinducers of apoptosis, there is growing evidence that these receptorsalso have non-apoptotic functions, including regulation of the adaptiveimmune response. Bamias et al., reported that TL1A is expressed bylamina propia dendritic cells and that it functions by increasing theproliferation of memory cells, but not naive CD4⁺ T cells, andsynergizes with IL-12 and/or low-dose stimulation of the T cell receptorto strongly enhance IFN-γ gene expression (Bamias G et al., (2006) Proc.Natl. Acad. Sci. USA, 103: 8441-8446). IFN-γ expression in the gut hasbeen considered a marker of inflammation and many strategies fortreating Crohn's disease rely on broad attempts to suppress theimmune-activated state. However, such approaches (steroid treatment andimmunosuppressive drugs) do not focus on the gut specifically andtherefore have their own complications. Targeted therapies based on theuse of antagonists of TNF-α were introduced with success in the 1990sand the results suggest that therapy directed specifically against TL1Aor its receptor may provide an alternative targeted therapy for thisdebilitating disorder.

Current treatments for Crohn's disease include the anti-TNF-α monoclonalantibodies Infliximab (Remicade®; Centocor) and Adalimumab (Humira®;Abbott), as well as anti-inflammatories (e.g., sulfasalazine), cortisoneor steroids (e.g., prednisone), immune system suppressors (e.g.,6-mercaptopurine) and antibiotics. However, Infliximab is the onlytreatment option having a high degree of specificity compared to theother available treatments (Young H A et al., (2006), supra). AlthoughInfliximab is generally well tolerated it can cause a recurrence oftuberculosis infection, worsening of heart failure, demyelinatingdisease and an increased incidence of lymphoma.

Therefore there remains a need in the art for compositions that can beused in the treatment and diagnosis of diverse inflammatory and immunediseases and disorders.

SUMMARY OF THE INVENTION

The present disclosure relates generally to antibodies or fragmentsthereof that bind to TL1A, methods for their preparation and use,including methods for treating TL1A mediated disorders. The antibodiesor fragments thereof of the present invention that bind to TL1A exhibitnumerous desirable properties and may be useful for the treatment ofvarious diseases that include but are not limited to inflammatorydiseases and/or auto immune diseases, including inter alia inflammatorybowel diseases (e.g., ulcerative colitis and Crohn's disease),rheumatoid arthritis, multiple sclerosis (MS), atherosclerosis,transplant rejection, central nervous system injury, psoriasis,leukaemia or lymphoma (e.g., chronic lymphocytic leukaemia (CLL)),atherosclerosis, and lung and colon carcinomas. The antibodies orfragments thereof of the present invention that bind to TL1A exhibitnumerous desirable properties and may be useful for the treatment ofvarious diseases that include but are not limited to inflammatorydiseases and/or auto immune diseases, including inter alia inflammatorybowel diseases (e.g., ulcerative colitis and Crohn's disease),rheumatoid arthritis, multiple sclerosis (MS), atherosclerosis,transplant rejection, central nervous system injury, psoriasis,leukaemia or lymphoma (e.g., chronic lymphocytic leukaemia (CLL)),atherosclerosis, and lung and colon carcinomas, chronic obstructivepulmonary disease COPD, optic neuritis, age related maculardegeneration, systemic lupus erythematosus (SLE), sjogen's syndrome,sclerodefina, systemic sclerosis, chronic Kidney disease, liverfibrosis, tuberculosis, idiopathic pulmonary fibrosis, tuberculosisinduced lung fibrosis, retroperitoneal Fibrosis, pulmonary fibrosis,cystic fibrosis, endomyocardial fibrosis, atrial fibrosis, mediastinalfibrosis, myelofibrosis (bone marrow), retroperitoneal fibrosis,progressive massive fibrosis, pephrogenic systemic fibrosis,arthrofibrosis.

In one aspect, the present disclosure provides an antibody or fragmentthereof that binds to TL1A comprising a heavy chain CDR1 comprising theamino acid sequence of SEQ ID NO: 51, and/or a heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 52, and/or a heavychain CDR3 comprising the amino acid sequence of SEQ ID NO: 53; and/orcomprising a light chain CDR1 comprising the amino acid sequence of SEQID NO: 54, and/or a light chain CDR2 comprising the amino acid sequenceof SEQ ID NO: 55 and/or a light chain CDR3 comprising the amino acidsequence of SEQ ID NO: 56.

In a further aspect the present invention provides an antibody orfragment thereof that binds to TL1A comprising a heavy chain variableregion sequence comprising the amino acid sequence of SEQ ID NO: 1. In afurther aspect the present invention provides an antibody or fragmentthereof that binds to TL1A comprising a heavy chain variable frameworkregion that is the product of or derived from a human gene selected fromthe group consisting of: IGHV1-2*02 (SEQ ID NO: 3), IGHV1-2*04 (SEQ IDNO: 4), IGHV1-2*05 (SEQ ID NO: 5), IGHV1-2*01 (SEQ ID NO: 6), andIGHV1-46*01 (SEQ ID NO: 7).

In a further aspect the present invention provides an antibody orfragment thereof comprising a heavy chain variable framework region thatis the product of or derived from human gene IGHV1-2*02 (SEQ ID NO: 3)and wherein the heavy chain variable framework region comprises at leastone amino acid modification from the corresponding framework region ofthe light chain variable region of the corresponding murine antibody.

In a further aspect the present invention provides an antibody orfragment thereof comprising a heavy chain sequence comprising the aminoacid sequence of SEQ ID NO: 13 and wherein the heavy chain variableframework region comprises at least one amino acid modification from thecorresponding heavy chain variable framework region of the correspondingmurine antibody.

In a further aspect the present invention provides an antibody orfragment thereof that binds to TL1A comprising a light chain variableregion sequence comprising the amino acid sequence of SEQ ID NO: 2. In afurther aspect the present invention provides an antibody or fragmentthereof that binds to TL1A comprising a light chain variable frameworkregion that is the product of or derived from a human gene selected fromthe group consisting of: IGKV1-33*01 (SEQ ID NO: 8), IGKV1D-33*01 (SEQID NO: 9), IGKV1D-12*02 (SEQ ID NO: 10), IGKV1D-12*01 (SEQ ID NO: 11)and IGKV1-12*02 (SEQ ID NO: 12).

In a further aspect the present invention provides an antibody orfragment thereof comprising a light chain variable framework region thatis the product of or derived from human gene IGKV1-33*01 (SEQ ID NO: 8)and wherein the light chain variable framework region comprises at leastone amino acid modification from the corresponding framework region ofthe light chain variable region of the corresponding murine antibody.

In a further aspect the present invention provides an antibody orfragment thereof that binds to TL1A comprising a heavy chain sequenceselected from the group consisting of SEQ ID NOS: 16, 21, 22, 23 and 24.In a further aspect the present invention provides an antibody orfragment thereof that binds to TL1A comprising a light chain sequenceselected from the group consisting of SEQ ID NOS: 17 and 25.

In a further aspect the present invention provides an antibody orfragment thereof that binds to TL1A comprising:

(a) a heavy chain sequence comprising the amino acid sequence of SEQ IDNO: 22 or 24; and

(b) a light chain sequence comprising the amino acid sequence of SEQ IDNO: 17.

In a further aspect the present invention provides an antibody orfragment thereof that binds to TL1A comprising a heavy chain variableregion comprising the amino acid sequence selected from the groupconsisting of SEQ ID NOS: 13, 26, 27, 28 and 29. In a further aspect thepresent invention provides an antibody or fragment thereof that binds toTL1A comprising a light chain variable region comprising the amino acidsequence selected from the group consisting of SEQ ID NOS: 14 and 30.

In a further aspect the present invention provides an antibody orfragment thereof that binds to TL1A comprising:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 27 or 29; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 14.

In a further aspect the present invention provides an antibody orfragment thereof that binds to TL1A, wherein the antibody comprises ahuman IgG4 Fc region, wherein the antibody has no Fc-mediatedcytotoxicity activity. In a further aspect the present inventionprovides an antibody or fragment thereof that binds to TL1A, wherein theantibody comprises a human IGHG1 Fc region, wherein the antibody iscompetent for cytotoxicity mechanisms such as antibody dependentcellular cytotoxicity (ADCC). In a preferred aspect, the antibody orfragment thereof that binds to TL1A has a non fucosylated IGHG1 Fcregion and exhibits enhanced Fc-mediated cytotoxicity mechanisms such asADCC.

In another aspect, the present invention provides a cross-reactiveantibody of fragment thereof which binds to human TL1A and which alsobinds to murine, rat and cynomologous TL1A. By “cross-reactive antibody”is meant an antibody that binds to an antigen from one species, e.g.human, and which also binds to the corresponding antigen in a differentspecies, e.g. rat.

In another aspect, the disclosure of the present invention alsodescribes humanized antibodies or fragments thereof that bind with asimilar affinity to TL1A as the corresponding chimeric antibody e.g.retain at least 85% of the TL1A binding affinity (K_(D)) of thecorresponding chimeric antibody or have at least equivalent or higherTL1A binding affinity (K_(D)) when compared to the correspondingchimeric antibody. In a preferred aspect the humanised antibody orfragment thereof has approximately a three-fold higher TL1A bindingaffinity when compared to the corresponding chimeric antibody.

In a further aspect, the present invention also describes humanizedantibodies or fragments thereof that bind to hTL1A and inhibits theinteraction of hTL1A with both DR3 and DcR3.

The disclosure of the present invention also provides isolated nucleicacids encoding antibodies and fragments thereof that bind to TL1A,vectors and host cells comprising the nucleic acid or the vector.Compositions comprising the anti-TL1A antibody or fragment thereof and apharmaceutically acceptable carrier and immunoconjugates comprising theantibody or fragment thereof linked to a therapeutic agent are alsoprovided.

The present disclosure also provides methods for treating TL1A mediateddisorders. In one aspect, in an in vitro model of TL1A-induced IFNγsecretion by primed CD4 T cells, an anti-TL1A antibody or fragmentthereof efficiently suppressed the production of IFNγ induced by immunecomplex-stimulated monocytes. In another aspect, in an in vivo model ofallergic asthma, an anti-TL1A antibody reduced the number of eosinophilsin bronchoalveolar lavage fluid of asthmatic mice by approximately4-fold. In a further aspect, in an in vivo model of acute colitisinduced in mice with dextran sulphate sodium (DSS) and in rats bytrinitrobenzenesulfonic acid (TNBS), an anti-TL1A antibody was effectivein reducing the symptoms of disease.

The present disclosure also provides pharmaceutical compositionscomprising an anti-TL1A antibody or fragments thereof and a carrier,such as a diluent or excipient.

The present disclosure also provides kits and articles of manufacturecomprising the antibody or fragments thereof, a composition or animmunoconjugate for the treatment of a TL1A mediated disorder.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: These figures show the binding of hybridoma antibodiesto human TL1A-his (FIG. 1A) or the irrelevant protein-his (FIG. 1B),detected using an HRP-labelled anti-mouse IgG secondary antibody and TMBsubstrate. FIG. 1A displays the absorbance at 450 nm of an ELISA againstcoated human TL1A-his and FIG. 1B displays the absorbance at 450 nm ofan ELISA against an irrelevant protein-his.

FIG. 2: This figure shows the effect of purified hybridoma anti-TL1Aantibodies at three different concentrations in a blocking ELISA, wherethe binding of human TNFRSF25 to TL1A was evaluated in the presence ofthe antibodies shown in FIG. 2. Absorbance was read at 450 nm. mIgG:mouse IgG isotype control.

FIGS. 3A, 2B, and 3C: Parental 5G6 candidate blocks the effect ofsoluble and membrane bound TL1A produced by activated monocytes: FIG.3A: PBMCs from healthy donors were stimulated with immune complexes thenstained with fluorescent antibodies. The monocytes were gated based onlarge forward light scatter and high side scatter parameters. Thehistogram plot displays the PE fluorescence of monocytes gatedpopulation. The grey shaded histogram represents the isotype control andthe blank histogram represents the staining with anti TL1A. FIG. 3B: Thesupernatants of purified human monocytes from healthy donor PBMCsstimulated with immune complex were harvested and tested by ELISA forthe presence of sTL1A proteins. The graph shows the interpolated TL1Aconcentration measured in the supernatants of indicated conditions. ‘ICstim’ means immune complex stimulated. ‘NS’ means not stimulated. FIG.3C: Naïve CD4 T cells purified from healthy donor PBMCs were incubatedwith IL-12, IL-18 and IC stimulated autologous monocytes. The parentalchimeric 5G6 antibody was added at the concentrations indicated in thetable at the same time as the monocytes. NA indicates that no IL-12 andIL-18 was added. The supernatants of cultures were quantified by ELISA.The graph shows the interpolated IFN-γ concentration for each indicatedcondition.

FIG. 4: Parental 5G6 candidate binds to mouse, rat, cynomologus monkeyand human TL1A: The binding of 5G6 on the extracellular part of TL1Aprotein corresponding to human (homo sapiens), rat (ratus norvegicus),mouse (mus musculus) and cynomologus monkey (macaca fascicularis)sequences was determined by immunofluorescence. The graph shows theabsorbance at 450 nm according to the log of the concentration of 5G6used.

FIGS. 5A, 5B, 5C, and 5D: Humanized 5G6 antibodies block TL1A-inducedIFN-γ secretion by primed CD4 T cells: Naïve CD4 T cells were incubatedwith IL-12, IL-18 and recombinant soluble human TL1A and the humanized5G6 candidates (VH3/VL1 (FIG. 5A), VH4/VL1 (FIG. 5B), VH5/VL (FIG. 5C)and VH2/VL2 (FIG. 5D)) were added at the concentrations indicated in thetable. NA indicates that no IL-12 and IL-18 was added. The culturesupernatants were quantified by ELISA for concentration of IFN-γ. FIGS.5A-5D show the IFN-γ concentration for each culture condition. Eachgraph displays the result of one humanized 5G6 candidate.

FIG. 6: Humanized 5G6 antibody reduced the number of cells inbronchoalveolar lavage (BAL) fluid in a murine model of allergic asthma.Mice were treated with the humanized 5G6 candidate (VH5/VL1; format IgG4hinge stabilised) at 50 mg/kg or an equivalent amount of control humanIgG, or dexamethasone at 5 mg/kg (positive control), on day 28, 30 and33 following the induction of an immunological response induced byovalbumin challenge. The graph shows the number of eosinophils in BALfluid for each mouse and the average number of eosinophils for eachgroup was calculated. Standard deviation was calculated using a one wayANOVA * indicates p<0.05, and ** indicates p<0.01.

FIG. 7: Treatment by humanized 5G6 antibody ameliorates shortening ofcolon in a DSS-induced model of acute colitis. Mice were treated 3× weekwith 50 mg/kg of a humanized 5G6 antibody (VH5/VL1; format IgG4 hingestabilised) or an equivalent amount of isotype control, or cyclosporineat 5 mg/kg (positive control). The graph shows the entire colon lengthfor each mouse and the average length per group. Standard deviation wascalculated using a one way ANOVA * indicates p<0.05, ** indicates p<0.01and *** indicates p<0.001.

FIG. 8: Treatment by humanized 5G6 antibody ameliorates disease severityin a TNBS-induced model of acute colitis. Rats were treated i.p with asingle dose of humanized 5G6 antibody (50 mg/kg) or an equal amount ofisotype control, two hours after TNBS administration. Prednisolone wasadministered as a positive control. Disease severity was assessed usinga colonic score for adhesions, strictures, ulcers and wall thickness andthe average score is shown in the histograms. Standard deviation wascalculated using a Student's t-test and * indicates p<0.05.

FIG. 9: Binding of 5G6 to hTL1A is blocked by both hDcR3-Fc and hDR3-Fc.Histidine-tagged human TL1A was coated at 2 μg/ml on an ELISA plate andincubated with 20 μg/ml 5G6 in the presence of 10 μg/ml Fc fusions ofthe ectodomains of either human DcR3 (white bar), DR3 (black bar) or anirrelevant receptor (Ctrl-Fc, hatched bar) followed by detection withperoxidase-conjugated anti-human IgG (Fab specific).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to antibodies and fragments thereof thatbind to TL1A.

The term “TL1A” as used herein includes variants, isoforms, and specieshomologs of TL1A. Accordingly, antibodies of this disclosure may bind tohuman TLA1 and may cross-react with TL1A from species other than human,for example, mouse, rat or cynomologous monkey. In certain embodiments,the antibodies may be completely specific for one or more human TL1Aproteins and may not exhibit species or other types of non-humancross-reactivity. The complete amino acid sequence of an exemplary humanTL1A has Swiss-Prot accession number O95150 (TNFSF15_HUMAN; SEQ IDNO:38). TL1A is also known as TNFSF15; TNF-like protein 1A; VEGI;TNF-γβ. Human TL1A is designated GeneID: 9966 by Entrez Gene, and HGNC:11931 by HGNC. TL1A can be encoded by the gene designated TNFSF15/TL1A.The complete amino acid sequence of an exemplary murine TL1A hasSwiss-Prot accession number Q5UBV8 (TNFSF15_MOUSE; SEQ ID NO: 39).Murine TL1A is designated GeneID: 326623 by Entrez Gene. The completeamino acid sequence of an exemplary rat TL1A has Swiss-Prot accessionnumber Q8K3Y7 (TNFSF15_RAT; SEQ ID NO: 40). Rat TL1A is designatedGeneID: 252878 by Entrez Gene. The complete amino acid sequence of anexemplary cyno TL1A (macaca fascicularis) has SEQ ID NO: 41.

The use of “TL1A” herein encompasses all known or as yet undiscoveredalleles and polymorphic forms of TL1A, preferably human TL1A.

The term “antibody or fragment thereof that binds to TL1A” as usedherein includes antibodies or a fragment thereof that binds to TL1A e.g.human TL1A in isolated form, with an affinity (K_(D)) of 850 pM or less,preferably 700 nM or less, more preferably 300 nM or less, morepreferably 260 nM or less, even more preferably 250 nM or less.

The tern “antibody or fragment thereof that binds to TL1A” includesantibodies or antigenic binding fragments thereof.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragments or single chains thereof. An “antibody”refers to a glycoprotein comprising at least two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, or an antigenbinding fragment thereof. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as VL) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR) withare hypervariable in sequence and/or involved in antigen recognitionand/or usually form structurally defined loops, interspersed withregions that are more conserved, termed framework regions (FR or FW).Each VH and VL is composed of three CDRs and four FWs, arranged fromamino-terminus to carboxy-terminus in the following order: FW1, CDR1,FW2, CDR2, FW3, CDR3, FW4. The amino acid sequences of FW1, FW2, FW3,and FW4 all together constitute the “non-CDR region” or “non-extendedCDR region” of VH or VL as referred to herein.

The term “heavy chain variable framework region” as referred herein maycomprise one or more (e.g., one, two, three and/or four) heavy chainframework region sequences (e.g., framework 1 (FW1), framework 2 (FW2),framework 3 (FW3) and/or framework 4 (FW4)). Preferably the heavy chainvariable region framework comprises FW1, FW2 and/or FW3, more preferablyFW1, FW2 and FW3. The term “light chain variable framework region” asreferred herein may comprise one or more (e.g., one, two, three and/orfour) light chain framework region sequences (e.g., framework 1 (FW1),framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4)).Preferably the light chain variable region framework comprises FW1, FW2and/or FW3, more preferably FW1, FW2 and FW3.

The variable regions of the heavy and light chains contain a bindingdomain that interacts with an antigen. The constant regions of theantibodies may mediate the binding of the immunoglobulin to host tissuesor factors, including various cells of the immune system (e.g., effectorcells) and the first component (C1q) of the classical complement system.

Antibodies are grouped into classes, also referred to as isotypes, asdetermined genetically by the constant region. Human constant lightchains are classified as kappa (CK) and lambda (Cλ) light chains. Heavychains are classified as mu (μ), delta (δ), gamma (γ), alpha (α), orepsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA,and IgE, respectively. Thus, “isotype” as used herein is meant any ofthe classes and/or subclasses of immunoglobulins defined by the chemicaland antigenic characteristics of their constant regions. The known humanimmunoglobulin isotypes are IgG1 (IGHG1), IgG2 (IGHG2), IgG3 (IGHG3),IgG4 (IGHG4), IgA1 (IGHA1), IgA2 (IGHA2), IgM (IGHM), IgD (IGHD), andIgE (IGHE). The so-called human immunoglobulin pseudo-gamma IGHGP generepresents an additional human immunoglobulin heavy constant region genewhich has been sequenced but does not encode a protein due to an alteredswitch region (Bensmana M et al., (1988) Nucleic Acids Res. 16(7):3108). In spite of having an altered switch region, the humanimmunoglobulin pseudo-gamma IGHGP gene has open reading frames for allheavy constant domains (CH1-CH3) and hinge. All open reading frames forits heavy constant domains encode protein domains which align well withall human immunoglobulin constant domains with the predicted structuralfeatures. This additional pseudo-gamma isotype is referred herein asIgGP or IGHGP. Other pseudo immunoglobulin genes have been reported suchas the human immunoglobulin heavy constant domain epsilon P1 and P2pseudo-genes (IGHEP1 and IGHEP2). The IgG class is the most commonlyused for therapeutic purposes. In humans this class comprises subclassesIgG1, IgG2, IgG3 and IgG4. In mice this class comprises subclasses IgG1,IgG2a, IgG2b, IgG2c and IgG3.

The term “murine antibody” as used herein includes antibodies in whichthe variable region sequences and the constant region sequences arederived from a mouse.

The term “chimeric antibody” as used herein includes antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

The term “humanized antibody” or “humanized anti-TL1A antibody” as usedherein includes antibodies in which CDR sequences derived from thegermline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences. Additional framework regionmodifications may be made within the human framework sequences as wellas within the CDR sequences derived from the germline of anothermammalian species.

The term “neutralising antibody” includes an antibody that is capable ofinhibiting and/or neutralising the biological activity of TL1A, forexample by blocking binding or substantially reducing binding of TL1A toits receptor TNFRSF25/DR3 or the decoy receptor TNFRSF21/DR6 and thusinhibiting or reducing the signalisation pathway triggered by TL1Aand/or inhibiting or reducing a TL1A-mediated cell response like e.g.lymphocyte proliferation, cytokine expression, or lymphocyte survival.

The terms “antagonistic antibody” or “antagonist antibody” are usedherein equivalently and include an antibody that is capable ofinhibiting and/or neutralising the biological signalling activity ofTL1A, as described for a neutralising antibody supra.

The terms “agonistic antibody” or agonist antibody” are used hereinequivalently and include an antibody that is capable of activatingand/or enhancing the biological signalling activity of TL1A, for exampleby increasing binding of TL1A to its receptor TNFRSF25/DR3 or the decoyreceptor TNFRSF21/DR6 and thus activating or enhancing the signalisationpathway triggered by TL1A and/or activating or enhancing a TL1A-mediatedcell response like e.g. lymphocyte proliferation, cytokine expression,or lymphocyte survival.

The term “Fab” or “Fab region” as used herein includes the polypeptidesthat comprise the VH, CH1, VL, and CL immunoglobulin domains. Fab mayrefer to this region in isolation, or this region in the context of afull length antibody or antibody fragment.

The term “Fc” or “Fc region”, as used herein includes the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-teuninal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domains Cgamma 2 and C gamma 3 (Cγ2 and Cγ3) and the hinge between C gamma 1(Cγ1) and C gamma 2 (Cγ2). Although the boundaries of the Fc region mayvary, the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU numbering system. For human IgG1 the Fc region isherein defined to comprise residue P232 to its carboxyl-terminus,wherein the numbering is according to the EU numbering system (Edelman GM et al., (1969) Proc Natl Acad Sci USA, 63(1): 78-85). Fc may refer tothis region in isolation or this region in the context of an Fcpolypeptide, for example an antibody.

The term “hinge” or “hinge region” or “antibody hinge region” hereinincludes the flexible polypeptide comprising the amino acids between thefirst and second constant domains of an antibody. The “hinge region” asreferred to herein is a sequence region of 6-62 amino acids in length,only present in IgA, IgD and IgG, which encompasses the cysteineresidues that bridge the two heavy chains. Structurally, the IgG CH1domain ends at EU position 220, and the IgG CH2 domain begins at residueEU position 237. Thus for IgG the antibody hinge is herein defined toinclude positions 221 (D221 in IgG1) to 231 (A231 in IgG1), wherein thenumbering is according to the EU numbering system (Edelman G M et al.,supra).

The term “parent antibody” or “parent immunoglobulin” as used hereinincludes an unmodified antibody that is subsequently modified togenerate a variant. Said parent antibody may be a naturally occurringantibody, or a variant or engineered version of a naturally occurringantibody. Parent antibody may refer to the antibody itself, compositionsthat comprise the parent antibody, or the amino acid sequence thatencodes it. By “parent anti-TL1A antibody” as used herein is meant anantibody or immunoglobulin that binds TL1A and is modified to generate avariant. By “corresponding murine antibody” as used herein is meant amurine antibody or immunoglobulin that binds to TL1A and that can bemodified to generate a variant, specifically the murine antibody 5G6 asdisclosed herein. By “corresponding chimeric antibody” as used herein ismeant a chimeric antibody or immunoglobulin that binds to TL1A and thatcan be modified to generate a variant.

The term “variant antibody” or “antibody variant” as used hereinincludes an antibody sequence that differs from that of a parentantibody sequence by virtue of at least one amino acid modificationcompared to the parent. The variant antibody sequence herein willpreferably possess at least about 80%, most preferably at least about90%, more preferably at least about 95% amino acid sequence identitywith a parent antibody sequence. Antibody variant may refer to theantibody itself, compositions comprising the antibody variant, or theamino acid sequence that encodes it.

The teixn “identity” or “substantial identity” or “substantiallyidentical,” when referring to a nucleic acid or fragment thereof,indicates that, when optimally aligned with appropriate nucleotideinsertions or deletions with another nucleic acid (or its complementarystrand), there is nucleotide sequence identity in at least about 80%,and more preferably at least about 90%, 95%, 96%, 97%, 98% or 99% of thenucleotide bases, as measured by any well-known algorithm of sequenceidentity, such as FASTA, BLAST or GAP, as discussed below.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 80% sequence identity, even more preferably atleast 90%, 95%, 98% or 99% sequence identity. Preferably, residuepositions which are not identical differ by conservative amino acidsubstitutions.

The term “amino acid modification” herein includes an amino acidsubstitution, insertion, and/or deletion in a polypeptide sequence. By“amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with another amino acid. For example, thesubstitution R94K refers to a variant polypeptide, in this case a heavychain variable framework region variant, in which the arginine atposition 94 is replaced with a lysine. For the preceding example, 94Kindicates the substitution of position 94 with a lysine. For thepurposes herein, multiple substitutions are typically separated by aslash. For example, R94K/L78V refers to a double variant comprising thesubstitutions R94K and L78V. By “amino acid insertion” or “insertion” asused herein is meant the addition of an amino acid at a particularposition in a parent polypeptide sequence. For example, insert −94designates an insertion at position 94. By “amino acid deletion” or“deletion” as used herein is meant the removal of an amino acid at aparticular position in a parent polypeptide sequence. For example,R94—designates the deletion of arginine at position 94.

As used herein, the term “conservative modifications” or “conservativesequence modifications” is intended to refer to amino acid modificationsthat do not significantly affect or alter the binding characteristics ofthe antibody containing the amino acid sequence. Such conservativemodifications include amino acid substitutions, insertions anddeletions. Modifications can be introduced into an antibody of theinvention by standard techniques known in the art, such as site-directedmutagenesis and PCR-mediated mutagenesis.

Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within the CDR regions or within the frameworkregions of an antibody of the invention can be replaced with other aminoacid residues from the same side chain family and the altered antibody(variant antibody) can be tested for retained function.

The term “epitope” refers to a region of an antigen that is bound by anantibody. An epitope may be defined as structural or functional.Functional epitopes are generally a subset of structural epitopes andhave those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

For all human immunoglobulin heavy chain constant domains numbering isaccording to the “EU numbering system” (Edelman G M et al., (1969) ProcNatl Acad Sci USA, 63(1): 78-85).

For the human kappa immunoglobulin light chain constant domain (IGKC),numbering is according to the “EU numbering system” (Edelman G M et al.,supra).

For the human lambda immunoglobulin light chain constant domains (IGLC1,IGLC2, IGLC3, IGLC6, and IGLC7), numbering is according to the “Kabatnumbering system” (Kabat E A et al., (1991) Sequences of proteins ofimmunological interest. 5th Edition—US Department of Health and HumanServices, NIH publication no 91-3242) as described by Dariavach P etal., (1987) Proc Natl Acad Sci USA, 84(24): 9074-8 and Frangione B etal., (1985) Proc Natl Acad Sci USA, 82(10): 3415-9.

The term “variable domain” refers to the domains that mediatesantigen-binding and defines specificity of a particular antibody for aparticular antigen. In naturally occurring antibodies, theantigen-binding site consists of two variable domains that definespecificity: one located in the heavy chain (VH) and the other locatedin the light chain (VL). In some cases, specificity may exclusivelyreside in only one of the two domains as in single-domain antibodiesfrom heavy-chain antibodies found in camelids. The V regions are usuallyabout 110 amino acids long, and consist of relatively invariantstretches of amino acid sequence called framework regions (FRs) of 15-30amino acids separated by shorter regions of extreme variability called“hypervariable regions” that are 9-12 amino acids long. The variabledomains of native heavy and light chains comprise four FRs, largelyadopting a beat-sheet configuration, connected by three hypervariableregions, which form loops. The hypervariable regions in each chain areheld together in close proximity by FRs, and with the hypervariableregions from the other chain, contribute to the formation of the antigenbinding site of antibodies (see Kabat E A et al., supra). The term“hypervariable region” as used herein refers to the amino acid residuesof an antibody which are responsible for antigen binding. Thehypervariable region generally comprises amino acid residues from a“complementary determining region” or “CDR”, the latter being of highestsequence variability and/or involved in antigen recognition. For allvariable domains numbering is according to Kabat (Kabat E A et al.,supra).

A number of CDR definitions are in use and are encompassed herein. TheKabat definition is based on sequence variability and is the mostcommonly used (Kabat E A et al., supra). Chothia refers instead to thelocation of the structural loops (Chothia C & Lesk A M (1987) J. Mol.Biol. 196: 901-917). The AbM definition is a compromise between theKabat and the Chothia definitions and is used by Oxford Molecular's AbMantibody modelling software (Martin A C R et al., (1989) Proc. NatlAcad. Sci. USA, 86: 9268-72; Martin A C R et al., (1991) MethodsEnzymol. 203: 121-153; Pedersen J T et al., (1992) Immunomethods, 1:126-136; Rees A R et al., (1996) In Sternberg M. J. E. (ed.), ProteinStructure Prediction. Oxford University Press, Oxford, 141-172). Thecontact definition has been recently introduced (MacCallum R M et al.,(1996) J. Mol. Biol. 262: 732-745) and is based on an analysis of theavailable complex structures available in the Protein Databank. Thedefinition of the CDR by IMGT®, the international ImMunoGeneTicsinformation system® is based on the IMGT numbering for allimmunoglobulin and T cell receptor V-REGIONs of all species (IMGT®, theinternational ImMunoGeneTics information system®; Lefranc M P et al.,(1991) Nucleic Acids Res. 27(1): 209-12; Ruiz M et al., (2000) NucleicAcids Res. 28(1): 219-21; Lefranc M P (2001) Nucleic Acids Res. 29(1):207-9; Lefranc M P (2003) Nucleic Acids Res. 31(1): 307-10; Lefranc M Pet al., (2005) Dev. Comp. Immunol. 29(3): 185-203; Kaas Q et al., (2007)Briefings in Functional Genomics & Proteomics, 6(4): 253-64).

All Complementarity Determining Regions (CDRs) discussed in the presentinvention, are defined preferably according to IMGT®. The variabledomain residues for each of these CDRs are as follows (numberingaccording to Kabat E A, et al., supra): LCDR1: 27-32, LCDR2: 50-52,LCDR3: 89-97, HCDR1: 26-35, HCDR2: 51-57 and HCDR3: 93-102. The “non-CDRregion” of the VL region as used herein comprise the amino acidsequences: 1-26 (FR1), 33-49 (FR2), 53-88 (FR3), and 98- approximately107 (FR4). The “non-CDR region” of the VH region as used herein comprisethe amino acid sequences: 1-25 (FR1), 36-50 (FR2), 58-92 (FR3), and 103-approximately 113 (FR4).

The CDRs of the present invention may comprise “extended CDRs” which arebased on the aforementioned definitions and have variable domainresidues as follows: LCDR1: 24-36, LCDR2: 46-56, LCDR3:89-97, HCDR1:26-36, HCDR2:47-65, HCDR3: 93-102. These extended CDRs are numbered aswell according to Kabat et al., supra. The “non-extended CDR region” ofthe VL region as used herein comprise the amino acid sequences: 1-23(FR1), 37-45 (FR2), 57-88 (FR3), and 98- approximately 107 (FR4). The“non-extended CDR region” of the VH region as used herein comprise theamino acid sequences: 1-25 (FR1), 37-46 (FR2), 66-92 (FR3), and 103-approximately 113 (FR4).

The term “full length antibody” as used herein includes the structurethat constitutes the natural biological form of an antibody, includingvariable and constant regions. For example, in most mammals, includinghumans and mice, the full length antibody of the IgG class is a tetramerand consists of two identical pairs of two immunoglobulin chains, eachpair having one light and one heavy chain, each light chain comprisingimmunoglobulin domains VL and CL, and each heavy chain comprisingimmunoglobulin domains VH, CH1 (Cγ1), CH2 (Cγ2), and CH3 (Cγ3). In somemammals, for example in camels and llamas, IgG antibodies may consist ofonly two heavy chains, each heavy chain comprising a variable domainattached to the Fc region.

Antibody fragments include, but are not limited to, (i) the Fab fragmentconsisting of VL, VH, CL and CH1 domains, including Fab′ and Fab′-SH,(ii) the Fd fragment consisting of the VH and CH1 domains, (iii) the Fvfragment consisting of the VL and VH domains of a single antibody; (iv)the dAb fragment (Ward E S et al., (1989) Nature, 341: 544-546) whichconsists of a single variable, (v) F(ab′)2 fragments, a bivalentfragment comprising two linked Fab fragments (vi) single chain Fvmolecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird R E et al., (1988) Science 242: 423-426;Huston J S et al., (1988) Proc. Natl. Acad. Sci. USA, 85: 5879-83),(vii) bispecific single chain Fv dimers (PCT/US92/09965), (viii)“diabodies” or “triabodies”, multivalent or multispecific fragmentsconstructed by gene fusion (Tomlinson I & Hollinger P (2000) MethodsEnzymol. 326: 461-79; WO94/13804; Holliger P et al., (1993) Proc. Natl.Acad. Sci. USA, 90: 6444-48) and (ix) scFv genetically fused to the sameor a different antibody (Coloma M J & Morrison S L (1997) NatureBiotechnology, 15(2): 159-163).

The term “effector function” as used herein includes a biochemical eventthat results from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include FcγR-mediated effectorfunctions such as ADCC (antibody dependent cell-mediated cytotoxicity)and ADCP (antibody dependent cell-mediated phagocytosis), andcomplement-mediated effector functions such as CDC (complement dependentcytotoxicity). An effector function of an antibody may be altered byaltering, i.e. enhancing or reducing, preferably enhancing, the affinityof the antibody for an effector molecule such as an Fc receptor or acomplement component. Effector function may be determined using one ormore cell based or in vivo assays. Such assays often involve monitoringthe response of cells to the antibody, for example cell survival, celldeath, change in cellular morphology or transcriptional activation suchas cellular expression of a natural gene or reporter gene. For example,such assays may measure the ability of an antibody to elicit ADCC, ADCP,or CDC. For some assays additional cells or components, that is inaddition to the target cells, may need to be added, for example serumcomplement or effector cells such as peripheral blood monocytes (PBMCs),NK cells, macrophages, and the like. Enhanced effector function can bedetermined by comparing the effector function of an altered antibodywith a control antibody and detecting, for example, an increase in ADCC,ADCP or CDC measured by one of more of the aforementioned assays.

Binding affinity will generally be varied by modifying the effectormolecule binding site and in this case it is appropriate to locate thesite of interest and modify at least part of the site in a suitable way.It is also envisaged that an alteration in the binding site on theantibody for the effector molecule need not alter significantly theoverall binding affinity but may alter the geometry of the interactionrendering the effector mechanism ineffective as in non-productivebinding. It is further envisaged that an effector function may also bealtered by modifying a site not directly involved in effector moleculebinding, but otherwise involved in performance of the effector function.By altering an effector function of an antibody it may be possible tocontrol various aspects of the immune response, e.g. enhancing orsuppressing various reactions of the immune system, with possiblebeneficial effects in diagnosis and therapy.

As used herein, the term “TL1A-mediated disorder” includes conditionssuch as inflammatory diseases and/or auto immune diseases, includinginter alia inflammatory bowel diseases (e.g., ulcerative colitis andCrohn's disease), rheumatoid arthritis, multiple sclerosis (MS),atherosclerosis, transplant rejection, central nervous system injury,psoriasis, leukemia or lymphoma (e.g., chronic lymphocytic leukemia(CLL)), atherosclerosis, and lung and colon carcinomas, chronicobstructive pulmonary disease COPD, optic neuritis, age related maculardegeneration, systemic lupus erythematosus (SLE), sjogen's syndrome,scleroderma, systemic sclerosis, chronic Kidney disease, liver fibrosis,tuberculosis, idiopathic pulmonary fibrosis, tuberculosis induced lungfibrosis, retroperitoneal Fibrosis, pulmonary fibrosis, cystic fibrosis,endomyocardial fibrosis, atrial fibrosis, mediastinal fibrosis,myelofibrosis (bone marrow), retroperitoneal fibrosis, progressivemassive fibrosis, pephrogenic systemic fibrosis, arthrofibrosis.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc. Preferably thesubject is human.

Anti-TL1A Antibodies

In a first aspect the present invention provides an antibody or fragmentthereof that binds to TL1A comprising a heavy chain CDR1 comprising theamino acid sequence of SEQ ID NO: 51, and/or a heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 52, and/or a heavychain CDR3 comprising the amino acid sequence of SEQ ID NO: 53; and/orcomprising a light chain CDR1 comprising the amino acid sequence of SEQID NO: 54, and/or a light chain CDR2 comprising the amino acid sequenceof SEQ ID NO: 55 and/or a light chain CDR3 comprising the amino acidsequence of SEQ ID NO: 56.

In some embodiments the antibody or fragment thereof that binds to TL1Acomprises an extended heavy chain CDR1 comprising the amino acidsequence of SEQ ID NO: 57, and/or an extended heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 58, and/or an extendedheavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 59;and/or comprises an extended light chain CDR1 comprising the amino acidsequence of SEQ ID NO: 60, and/or an extended light chain CDR2comprising the amino acid sequence of SEQ ID NO: 61 and/or an extendedlight chain CDR3 comprising the amino acid sequence of SEQ ID NO: 62.

Preferably the antibody or fragment thereof that binds to TL1A andcomprises a heavy chain CDR1 comprising the amino acid sequence of SEQID NO: 51, a heavy chain CDR2 comprising the amino acid sequence of SEQID NO: 52, and a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 53 and/or a light chain CDR1 comprising the amino acidsequence of SEQ ID NO: 54, a light chain CDR2 comprising the amino acidsequence of SEQ ID NO: 55 and a light chain CDR3 comprising the aminoacid sequence of SEQ ID NO: 56. More preferably the antibody or fragmentthereof that binds to TL1A comprises a heavy chain CDR1 comprising theamino acid sequence of SEQ ID NO: 51, a heavy chain CDR2 comprising theamino acid sequence of SEQ ID NO: 52, and a heavy chain CDR3 comprisingthe amino acid sequence of SEQ ID NO: 53 and a light chain CDR1comprising the amino acid sequence of SEQ ID NO: 54, a light chain CDR2comprising the amino acid sequence of SEQ ID NO: 55 and a light chainCDR3 comprising the amino acid sequence of SEQ ID NO: 56. Preferably,the antibody or fragment thereof binds to human TL1A and is crossreactive with murine, rat and cyno TL1A.

It is well known in the art that the CDR3 domain, independently from theCDR1 and/or CDR2 domain(s), alone can determine the binding specificityof an antibody for a cognate antigen and that multiple antibodies canpredictably be generated having the same binding specificity based on acommon CDR3 sequence. See, for example, Klimka A et al., (2000) Br. J.Cancer, 83(2): 252-260 (describing the production of a humanizedanti-CD30 antibody using only the heavy chain variable domain CDR3 ofmurine anti-CD30 antibody Ki-4); Beiboer S H et al., (2000) J. Mol.Biol. 296: 833-849 (describing recombinant epithelial glycoprotein-2(EGP-2) antibodies using only the heavy chain CDR3 sequence of theparental murine MOC-31 anti-EGP-2 antibody); Rader C et al., (1998)Proc. Natl. Acad. Sci USA, 95: 8910-8915 (describing a panel ofhumanized anti-integrin avβ3 antibodies using a heavy and light chainvariable CDR3 domain of a murine anti-integrin avβ3 antibody LM609wherein each member antibody comprises a distinct sequence outside theCDR3 domain and capable of binding the same epitope as the parentalmurine antibody with affinities as high or higher than the parentalmurine antibody); Barbas C et al., (1994) J. Am. Chem. Soc. 116: 2161-62(disclosing that the CDR3 domain provides the most significantcontribution to antigen binding).

Accordingly, the present invention provides antibodies and fragmentsthereof that bind to TL1A comprising one or more heavy and/or lightchain CDR3 domains, in particular comprising heavy chain CDR3 comprisingthe amino acid sequence of SEQ ID NO: 53 and/or light chain CDR3comprising the amino acid sequence of SEQ ID NO: 56, wherein theantibody is capable of binding to TL1A. Within some embodiments, suchinventive antibodies comprising one or more heavy and/or light chainCDR3 domain from a non-human antibody (a) are capable of competing forbinding with; (b) retain the functional characteristics; (c) bind to thesame epitope; and/or (d) have a similar binding affinity as thecorresponding parental non-human e.g. murine antibody.

In further aspect the antibody or fragment thereof comprises a heavychain variable region sequence comprising the amino acid sequence of SEQID NO: 1. In further aspect the antibody or fragment thereof comprises anon-CDR region of a heavy chain variable region sequence which is atleast 80% identical to the non-CDR region of the heavy chain variableregion sequence of SEQ ID NO: 1.

In a further aspect the present invention provides an antibody orfragment thereof that binds to TL1A comprising a heavy chain variableregion sequence comprising the amino acid sequence selected from SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.

In a further aspect the present invention provides an antibody orfragment thereof that binds to TL1A comprising a heavy chain variableregion sequence comprising the amino acid sequence of SEQ ID NO: 29. Inanother aspect the present invention provides an antibody or fragmentthereof that binds to TL1A comprising a light chain variable regionsequence comprising the amino acid sequence of SEQ ID NO: 14. In someembodiments the antibody or fragment thereof that binds to TL1Acomprises a heavy chain variable region sequence comprising the aminoacid sequence of SEQ ID NO: 29 and a light chain variable regionsequence comprising the amino acid sequence of SEQ ID NO: 14.Preferably, the antibody or fragment thereof binds to human TL1A and iscross reactive with murine, rat and cyno TL1A.

In another aspect the present invention provides variants of an antibodyor fragment thereof that binds to TL1A. Thus the present inventionprovides antibodies or fragments thereof that have an amino acidsequence of the non-CDR regions of the heavy and/or light chain variableregion sequence which is at least 80% identical (having at least 80%amino acid sequence identity) to the amino acid sequence of the non-CDRregions of the heavy and/or light chain variable region sequence of theparent antibody of either the heavy or the light chain e.g. of eitherthe heavy and light variable region sequences as in SEQ ID NO: 13, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 14,respectively. As well antibodies or fragments thereof that have an aminoacid sequence of the non-extended CDR regions of the heavy and/or lightchain variable region sequence which is at least 80% identical to theamino acid sequence of the non-extended CDR regions of the heavy and/orlight chain variable region sequence of the parent antibody of eitherthe heavy or the light chain are provided, by the present invention.Preferably the amino acid sequence identity of the non-CDR regions or ofthe non-extended CDR regions of the heavy and/or light chain variableregion sequence is at least 85%, more preferably at least 90%, and mostpreferably at least 95%, in particular 96%, more particular 97%, evenmore particular 98%, most particular 99%, including for example, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, and 100%. Identity or homology with respect toan amino acid sequence is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical with the antibodyor fragment thereof that binds to TL1A, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. Thus sequence identity can be determined by standard methodsthat are commonly used to compare the similarity in position of theamino acids of two polypeptides. Using a computer program such as BLASTor FASTA, two polypeptides are aligned for optimal matching of theirrespective amino acids (either along the full length of one or bothsequences or along a pre-determined portion of one or both sequences).The programs provide a default opening penalty and a default gappenalty, and a scoring matrix such as PAM250 (a standard scoring matrix;see Dayhoff M O et al., (1978) in Atlas of Protein Sequence andStructure, vol 5, supp. 3) can be used in conjunction with the computerprogram. For example, the percent identity can be calculated as: thetotal number of identical matches multiplied by 100 and then divided bythe sum of the length of the longer sequence within the matched span andthe number of gaps introduced into the longer sequences in order toalign the two sequences.

In some embodiments the present disclosure thus provides an antibody orfragment thereof that binds to TL1A, wherein the antibody or fragmentthereof comprises a heavy chain variable framework region sequence whichis at least 65% identical to the framework region sequence of SEQ IDNOS: 3, 4, 5, 6 or 7 and/or a light chain variable framework regionsequence which is at least 75% identical to the framework regionsequence of SEQ ID NOS: 8, 9, 10, 11 and 12. In some embodiments thepresent disclosure provides an antibody or fragment thereof that bindsto TL1A, wherein the antibody or fragment thereof comprises a heavychain variable framework region sequence which is at least 69% identicalto the framework region sequence of SEQ ID NO: 3 and/or a light chainvariable framework region sequence which is at least 80% identical tothe framework region sequence of SEQ ID NO: 8.

In another aspect the present invention provides an antibody or fragmentthereof that binds to TL1A comprising the heavy and or light chain CDRsas described supra and further comprising a heavy chain variableframework region that is the product of or derived from a human geneselected from the group consisting of IGHV1-2*02 (SEQ ID NO: 3),IGHV1-2*04 (SEQ ID NO: 4), IGHV1-2*05 (SEQ ID NO: 5), IGHV1-2*01 (SEQ IDNO: 6), and IGHV1-46*01 (SEQ ID NO: 7), preferably a heavy chainvariable framework region that is the product of or derived from humangene IGHV1-2*01 (SEQ ID NO: 3). The heavy chain variable frameworkregion may comprise one or more (e.g., one, two, three and/or four)heavy chain framework region sequences (e.g., framework 1 (FW1),framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4)) presentin the product of or derived from those human genes. Preferably theheavy chain variable region framework comprises FW1, FW2 and/or FW3,more preferably FW1, FW2 and FW3 present in the product of or derivedfrom a human gene selected from the group consisting of IGHV1-2*02 (SEQID NO: 3), IGHV1-2*04 (SEQ ID NO: 4), IGHV1-2*05 (SEQ ID NO: 5),IGHV1-2*01 (SEQ ID NO: 6), and IGHV1-46*01 (SEQ ID NO: 7). Heavy chainframework region sequences as used herein include FW1 (position 1 toposition 25), FW2 (position 36 to position 49), FW3 (position 66 toposition 94) and FW 4 (position 103 to position 113), wherein the aminoacid position is indicated utilizing the numbering system set forth inKabat.

In some embodiments the present disclosure provides an antibody orfragment thereof, wherein the antibody or fragment thereof comprises aheavy chain variable framework region that is the product of or derivedfrom human gene IGHV1-2*01 (SEQ ID NO: 3) and wherein the heavy chainvariable framework region comprises at least one amino acid modificationfrom the corresponding heavy chain variable framework region of thecorresponding murine antibody.

In some embodiments the present disclosure provides an antibody orfragment thereof comprising a heavy chain sequence comprising the aminoacid sequence of SEQ ID NO: 16 and wherein the heavy chain variableframework region comprises at least one amino acid modification from thecorresponding heavy chain variable framework region of the correspondingmurine antibody.

Preferably the amino acid modification comprises an amino acidsubstitution at amino acid position selected from the group consistingof 37, 48, 50, 67, 69, 71 and 75, more preferably at amino acidpositions selected from the group consisting of 37, 48, 50, 67 and 71,most preferred at amino acid position 37, wherein the amino acidposition of each group member is indicated according to the Kabatnumbering. Specifically the amino acid modification comprises an aminoacid substitution selected from the group consisting of 37A, 48I, 50E,67A, 69L, 71V and 75S, preferably an amino acid substitution selectedfrom the group consisting of V37A, M48I, W50E, V67A, M69L, R71V andI75S, whereas V37A is the most preferred amino acid substitution whereinthe amino acid position of each group member is indicated according tothe Kabat numbering.

In another aspect the present invention provides an antibody or fragmentthereof that binds to TL1A comprising a light chain variable frameworkregion that is the product of or derived from a human gene selected fromthe group consisting of IGKV1-33*01 (SEQ ID NO: 8), IGKV1D-33*01 (SEQ IDNO: 9), IGKV1D-12*02 (SEQ ID NO: 10), IGKV1D-12*01 (SEQ ID NO: 11), andIGKV1-12*02 (SEQ ID NO: 12), preferably a light chain variable frameworkregion that is the product of or derived from human gene IGKV1-33*01(SEQ ID NO: 8). The light chain variable region framework region maycomprise one or more (e.g., one, two, three and/or four) light chainframework region sequences (e.g., framework 1 (FW1), framework 2 (FW2),framework 3 (FW3) and/or framework 4 (FW4)) present in the product of orderived from those human genes. Preferably the light chain variableregion framework comprises FW1, FW2 and/or FW3, more preferably FW1, FW2and FW3 present in the product of or derived from a human gene selectedfrom the group consisting of IGKV1-33*01 (SEQ ID NO: 8), IGKV1D-33*01(SEQ ID NO: 9), IGKV1D-12*02 (SEQ ID NO: 10), IGKV1D-12*01 (SEQ ID NO:11), and IGKV1-12*02 (SEQ ID NO: 12). Light chain framework regionsequences as used herein include FW1 (position 1 to position 23), FW2(position 35 to position 49), FW3 (position 57 to position 88) and FW 4(position 98 to position 108), wherein the amino acid position isindicated utilizing the numbering system set forth in Kabat.

In some embodiments the present disclosure provides an antibody orfragment thereof comprising a light chain variable framework region thatis the product of or derived from human gene IGKV1-33*01 (SEQ ID NO: 8)and wherein the light chain variable framework region comprises at leastone amino acid modification from the corresponding framework region ofthe light chain variable region of the corresponding murine antibody.

In further aspect the antibody or fragment thereof comprises a lightchain variable region sequence comprising the amino acid sequence of SEQID NO: 2. In further aspect the antibody or fragment thereof comprises anon-CDR region of a light chain variable region sequence which is atleast 80% identical to the non-CDR region of the heavy chain variableregion sequence of SEQ ID NO: 2.

In some embodiments the present disclosure provides an antibody orfragment thereof comprising a light chain sequence comprising the aminoacid sequence selected from SEQ ID NO: 17 and SEQ ID NO: 25.

In some embodiments the present disclosure provides an antibody orfragment thereof comprising a light chain sequence comprising the aminoacid sequence of SEQ ID NO: 17. Alternatively, the light chain variableframework region of the light chain sequence comprises at least oneamino acid modification from the corresponding light chain variableframework region of the corresponding murine antibody.

The amino acid modification may comprise an amino acid substitution atan amino acid position selected from the group consisting of 5 and 34,wherein the amino acid position of each group member is indicatedaccording to the Kabat numbering. Specifically the amino acidmodification comprises an amino acid substitution selected from thegroup consisting of 5N, and 34S, preferably T5N and N34S, wherein theamino acid position of each group member is indicated according to theKabat numbering. Particularly preferred is a light chain sequencecomprising the amino acid sequence of SEQ ID NO: 17, without any aminoacid modifications.

In some embodiments the antibody or fragment thereof that binds to TL1Acomprises a heavy chain variable framework region that is the product ofor derived from a human gene selected from the group consisting ofIGHV1-2*02 (SEQ ID NO: 3), IGHV 1-2*04 (SEQ ID NO: 4), IGHV1-2*05 (SEQID NO: 5), IGHV1-2*01 (SEQ ID NO: 6), and IGHV1-46*01 (SEQ ID NO: 7) anda light chain variable framework region that is the product of orderived from a human gene selected from the group consisting ofIGKV1-33*01 (SEQ ID NO: 8), IGKV1D-33*01 (SEQ ID NO: 9), IGKV1D-12*02(SEQ ID NO: 10), IGKV1D-12*01 (SEQ ID NO: 11), and IGKV1-12*02 (SEQ IDNO: 12), preferably a heavy chain variable framework region that is theproduct of or derived from human gene IGHV1-2*02 (SEQ ID NO: 3), and alight chain variable framework region that is the product of or derivedfrom human gene IGKV1-33*01 (SEQ ID NO: 8). As well combinations ofheavy chain variable framework regions which are present in the productof or derived from different human genes mentioned supra and/or of lightchain variable region framework regions which are present in the productof or derived from different human genes mentioned supra are encompassedby the present invention.

Germline DNA sequences for human heavy and light chain variable regiongenes can be found in the “VBase” human germline sequence database(available on the Internet at www.mrccpe.cam.ac.uk/vbase), as well as inKabat E A et al., supra; Tomlinson I M et al., (1992) J. Mol. Biol. 227:776-798 and Cox J P L et al., (1994) Eur. J. Immunol. 24: 827-836. Asanother example, the germline DNA sequences for human heavy and lightchain variable region genes can be found in the Genbank database.

In another aspect, the present disclosure also provides an antibody orfragment thereof that binds to TL1A, wherein at least one of the heavychain CDRs and/or at least one of the light chain CDRs comprises atleast one amino acid modification. Site-directed mutagenesis orPCR-mediated mutagenesis can be performed to introduce themodification(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays.Preferably conservative modifications are introduced. Themodification(s) may be amino acid substitutions, additions or deletions,but are preferably substitutions. Typically, no more than five,preferably no more than four, more preferably no more than three, evenmore preferably no more than two, most preferably no more than one aminoacid modifications are performed within a CDR region.

In certain embodiments, framework sequences can be used to engineervariable regions to produce variant antibodies. Variant antibodies ofthe invention include those in which modifications have been made toframework residues within VH and/or VK, e.g. to improve the propertiesof the antibody. Typically such framework modifications are made todecrease the immunogenicity of the antibody. For example, one approachis to “backmutate” one or more framework residues to the correspondingmurine sequence or to “backmutate” one or more framework residues to acorresponding germline sequence.

Thus in a further aspect the present disclosure provides an antibody orfragment thereof that binds to TL1A, wherein at least one of theframework region sequences of the heavy chain variable region of theantibody or fragment thereof comprises at least one amino acidmodification from the corresponding framework region of the heavy chainvariable region of the corresponding murine antibody. Preferably theamino acid modification is an amino acid substitution. Typically, nomore than seven, preferably no more than six, preferably no more thanfive, preferably no more than four, more preferably no more than three,even more preferably no more than two, most preferably no more than oneamino acid modifications are perfotined within a framework region. Insome embodiments the present disclosure provides an antibody or fragmentthereof that binds to TL1A, wherein the amino acid modification of theframework regions of the heavy chain variable region comprise an aminoacid substitution at amino acid position selected from the groupconsisting of 37, 48, 50, 67, 69, 71, and 75 and wherein the amino acidposition of each group member is indicated according to the Kabatnumbering. Preferred amino acid substitution of the framework regions ofthe heavy chain variable region are at amino acid positions selectedfrom the group consisting of 37, 48, 50, 67 and 71. More preferred aminoacid substitutions of the framework regions of the heavy chain variableregion are selected from the group consisting of V37A, M48I, W50E, V67A,M69L, R71V and I75S, whereas V37A is the most preferred amino acidsubstitution of the framework regions of the heavy chain variableregion.

The present disclosure also provides an antibody or fragment thereofthat binds to TL1A, wherein at least one of the framework regionsequences of the light chain variable region of the antibody or fragmentthereof may comprise at least one amino acid modification from thecorresponding framework region of the light chain variable region of thecorresponding murine antibody. Preferably the amino acid modification isan amino acid substitution. Typically, no more than two, more preferablyno more than one and most preferably, no amino acid modifications areperformed within a framework region. In some embodiments the presentdisclosure provides an antibody or fragment thereof, wherein the aminoacid modification of the framework regions of the light chain variableregion sequence comprises an amino acid substitution at amino acidposition selected from the group consisting of 5 and 34. The amino acidmodifications of the framework regions of the light chain variableregion sequence comprise a substitution selected from the groupconsisting of a 5N and 34S, preferably T5N and N34S and wherein theamino acid position of each group member is indicated according to theKabat numbering. In some embodiments the antibody or fragment thereof ofthe present invention may comprise amino acid modifications of theframework regions of the heavy chain variable region sequence as set outabove and amino acid modifications of the framework regions of the lightchain variable region sequence as set out above.

The present disclosure also provides an antibody or fragment thereofthat binds to TL1A that comprises a heavy chain variable region selectedfrom the group consisting of SEQ ID NOS: 13, 26, 27, 28 and 29,preferably selected from the group consisting of SEQ ID NOS: 26, 27, 28and 29, more preferably from the group consisting of SEQ ID NOS: 27, 28and 29 and even more preferably from the group consisting of SEQ ID NOS:27 and 29. The present disclosure also provides an antibody or fragmentthereof that binds to TL1A that comprises a light chain variable regionselected from the group consisting of SEQ ID NOS: 14 and 30, morepreferably SEQ ID NO: 14. In some embodiments the antibody or fragmentthereof that binds to TL1A comprises a heavy chain variable regionselected from the group consisting of SEQ ID NOS: 26, 27, 28 and 29, anda light chain variable region selected from the group consisting of SEQID NOS: 14 and 30. Given that each of these heavy and light chainvariable region sequences can bind to TL1A, the heavy and light chainvariable region sequences can be “mixed and matched” to create anti-TL1Abinding molecules of the invention. TL1A binding of such “mixed andmatched” antibodies can be tested using the binding assays describede.g. in the Examples.

In some embodiments the antibody or fragment thereof that binds to TL1Acomprises a heavy chain variable region selected from the groupconsisting of SEQ ID NOS: 27 and 29, and a light chain variable regionselected from the group consisting of SEQ ID NOS: 14 and 30. In morepreferred embodiments the antibody or fragment thereof that binds toTL1A comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 27 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 14 or a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 29 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:14. Most preferred is an antibody or fragment thereof that binds to TL1Acomprising a heavy chain variable region selected from the groupconsisting of SEQ ID NOS: 27 and 29, and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 14.

The present disclosure also provides an antibody or fragment thereofthat binds to TL1A that comprises a heavy chain sequence selected fromthe group consisting of SEQ ID NOS: 16, 21, 22, 23 and 24, preferablyselected from the group consisting of SEQ ID NOS: 22, 23 and 24 and morepreferably from the group consisting of SEQ ID NOS: 22 and 24. Thepresent disclosure also provides an antibody or fragment thereof thatbinds to TL1A that comprises a light chain sequence selected from thegroup consisting of SEQ ID NOS: 17 and 25, more preferably SEQ ID NO:17. In some embodiments the antibody or fragment thereof that binds toTL1A comprises a heavy chain sequence selected from the group consistingof SEQ ID NOS: 21, 22, 23 and 24, and a light chain sequence selectedfrom the group consisting of SEQ ID NOS: 17 and 25. Given that each ofthese heavy and light chain variable region sequences can bind to TL1A,the heavy and light chain variable region sequences can be “mixed andmatched” to create anti-TL1A binding molecules of the invention. TL1Abinding of such “mixed and matched” antibodies can be tested using thebinding assays described e.g. in the Examples.

In some embodiments the antibody or fragment thereof that binds to TL1Acomprises a heavy chain sequence selected from the group consisting ofSEQ ID NOS: 22 and 24, and a light chain sequence selected from thegroup consisting of SEQ ID NOS: 17 and 25. In more preferred embodimentsthe antibody or fragment thereof that binds to TL1A comprises a heavychain sequence comprising the amino acid sequence of SEQ ID NO: 22 and alight chain sequence comprising the amino acid sequence of SEQ ID NO: 17or a heavy chain sequence comprising the amino acid sequence of SEQ IDNO: 24 and a light chain sequence comprising the amino acid sequence ofSEQ ID NO: 17. Most preferred is an antibody or fragment thereof thatbinds to TL1A comprising a heavy chain sequence selected from the groupconsisting of SEQ ID NOS: 22 and 24, and a light chain sequencecomprising the amino acid sequence of SEQ ID NO: 17.

In one embodiment of the present disclosure, the antibody or fragmentthereof is a humanized antibody. Preferably, the antibody or fragmentthereof is a humanized monoclonal antibody.

The present disclosure also provides a monovalent antibody or fragmentthereof that binds to TL1A, i.e. an antibody which consists of a singleantigen binding arm. The present disclosure also provides a fragment ofa antibody that binds to TL1A selected from the group consisting of Fab,Fab′, Fab′-SH, Fd, Fv, dAb , F(ab′)2, scFv, bispecific single chain Fvdimers, diabodies, triabodies and scFv genetically fused to the same ora different antibody. Preferred fragments are scFv, bispecific singlechain Fv dimers and diabodies. The present disclosure also provides afull length antibody that binds to TL1A.

The present disclosure also provides an antibody or fragment thereofthat binds to TL1A which further comprises a heavy and/or light constantregion in particular a human heavy and/or a human light constant region.Human heavy constant regions may be selected from the group of humanimmunoglobulins consisting of IgG1 (IGHG1), IgG2 (IGHG2), IgG3 (IGHG3),IgG4 (IGHG4), IgA1 (IGHA1), IgA2 (IGHA2), IgM (IGHM), IgD (IGHD), or IgE(IGHE), whereas the human heavy constant region IgG, in particular IgG1(IGHG1) is preferred. Human light constant region may be selected fromthe group of human immunoglobulins consisting of kappa or lambdaconstant regions, whereas human kappa constant region is preferred. In apreferred embodiment the antibody or fragment thereof that binds to TL1Acomprises a human IgG1 (IGHG1) heavy constant domain and a human lightkappa constant domain.

In addition or alternative to modifications made within the frameworkregions or CDR regions, antibodies of the invention may be engineered toinclude modifications within the Fc region, typically to alter one ormore functional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation. Each ofthese embodiments is described in further detail below. Modificationswithin the Fc region as outlined below are according to the EU numberingof residues in the Fc region. In one embodiment, the hinge region of CH1is modified such that the number of cysteine residues in the hingeregion is altered, e.g., increased or decreased. This approach isdescribed further in U.S. Pat. No. 5,677,425 by Bodmer et al. The numberof cysteine residues in the hinge region of CH1 is altered to, forexample, facilitate assembly of the light and heavy chains or toincrease or decrease the stability of the antibody. In anotherembodiment, the Fc hinge region of an antibody is mutated to decreasethe biological half life of the antibody. More specifically, one or moreamino acid mutations are introduced into the CH-2-CH3 domain interfaceregion of the Fc-hinge fragment such that the antibody has impairedStaphylococcal protein A (SpA) binding relative to native Fe-hingedomain SpA binding. This approach is described in further detail in U.S.Pat. No. 6,165,745 by Ward et al. In another embodiment, the antibody ismodified to increase its biological half life. Various approaches arepossible. For example, one or more of the following mutations can beintroduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375to Ward. Alternatively, to increase the biological half life, theantibody can be altered within the CH1 or CL region to contain a salvagereceptor binding epitope taken from two loops of a CH2 domain of an Fcregion of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022by Presta et al. In a further embodiment Fc region is altered byreplacing at least one amino acid residue with a different amino acidresidue to alter the effector function(s) of the antibody. For example,one or more amino acids selected from amino acid residues 234, 235, 236,237, 297, 318, 320 and 322 can be replaced with a different amino acidresidue such that the antibody has an altered affinity for an effectorligand but retains the antigen-binding ability of the parent antibody.The effector ligand to which affinity is altered can be, for example, anFc receptor or the C1 component of complement. This approach isdescribed in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260,both by Winter et al. In another example, one or more amino acidsselected from amino acid residues 329, 331 and 322 can be replaced witha different amino acid residue such that the antibody has altered C1qbinding and/or reduced or abolished complement dependent cytotoxicity(CDC). This approach is described in further detail in U.S. Pat. No.6,194,551 by Idusogie et al. In another example, one or more amino acidresidues within amino acid positions 231 to 238 in the N-terminal regionof the CH2 domain are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO94/29351 by Bodmer et al. In yet another example, the Fcregion is modified to increase the ability of the antibody to mediateantibody dependent cellular cytotoxicity (ADCC) and/or to increase theaffinity of the antibody for an Fcγ receptor by modifying one or moreamino acids at the following positions: 238, 239, 248, 249, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309,312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337,338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430,434, 435, 437, 438 or 439. This approach is described further in PCTPublication WO00/42072 by Presta.

The present disclosure also provides an antibody or fragment thereofthat binds to TL1A comprising human heavy and/or light constant regions,wherein the human heavy constant region comprises an isotypic variantcomprising the CH1 region, the hinge region, the CH2 region and CH3region from human IgG4 (IGHG4) and wherein the hinge region comprises asubstitution of serine at position 228 to proline. Preferably theantibody comprising the isotypic variant is a full length antibody. Aparticular preferred antibody or fragment thereof that binds to TL1Acomprising an isotypic variant comprising the CH1 from human IgG4(IGHG4), the hinge from human IgG4 (IGHG4), having S228P substitutionand the CH2 and CH3 from human IgG4 (IGHG4). It has been found that theisotypic variant exhibits no Fc-mediated cytotoxicity mechanisms such asADCC compared to an antibody or fragment thereof that binds to TL1Awhich comprises a human heavy constant region from human IgG1 (IGHG1)(which is usually a native human IgG1), i.e. as compared to an antibodyor fragment thereof that binds to TL1A that only differs from theisotypic variant with regard to the modified heavy constant region.

The present disclosure also provides an antibody or fragment thereofthat binds to TL1A which comprises a human IgG Fc region, wherein themature core carbohydrate structure attached to the human IgG Fc regionlacks fucose (referred herein alternatively as “non fucosylated”). Theterm “mature core carbohydrate structure” as used herein includes aprocessed core carbohydrate structure attached to an Fc region whichgenerally consists of the carbohydrate structure GlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc)₂ typical of biantennaryoligosaccharides represented schematically below:

This term specifically includes G-1 forms of the core maturecarbohydrate structure lacking a β1,2 GlcNAc residue. Preferably,however, the core carbohydrate structure includes both β1,2 GlcNAcresidues. The mature core carbohydrate structure herein generally is nothypermannosylated. The mature core carbohydrate structure is attached tothe Fc region of the glycoprotein, generally via N-linkage to Asn297 ofa CH2 domain of the Fc region.

Preferably the antibody comprises a human IgG1 (IGHG1) Fc region,wherein the mature core carbohydrate structure attached to the humanIgG1 (IGHG1) Fc region lacks fucose. More preferred is a full-lengthantibody comprising a human IgG1 (IGHG1) Fc region, wherein the maturecore carbohydrate structure attached to the human IgG1 (IGHG1) Fc regionlacks fucose. It is known from WO03/035835 that lack of fucose in themature core carbohydrate structure attached to the human IgG Fc regionmay enhance ADCC. Thus in a further embodiment the antibody or fragmentthereof of the present disclosure comprises a human IgG1 (IGHG1) Fcregion, wherein the mature core carbohydrate structure attached to thehuman IgG1 (IGHG1) Fe region lacks fucose, whereas the antibody lackingfucose exhibits enhanced ADCC compared to the parent antibody orfragment thereof not lacking fucose. Methods to generate antibodieswhich lack fucose are, for example (a) use of an engineered or mutanthost cell that is deficient in fucose metabolism such that it has areduced ability (or is unable to) fucosylate proteins expressed therein;(b) culturing cells under conditions which prevent or reducefucosylation; (c) post-translational removal of fucose (e. g. with afucosidase enzyme); (d) post-translational addition of the desiredcarbohydrate, e. g. after recombinant expression of a non-glycosylatedglycoprotein; or (e) purification of the glycoprotein so as to selectfor product which is not fucosylated. Preferably used are methodsdescribed in Example 14 of WO10/095031 e,g. methods described inLongmore et al., (1982) Carbohydr. Res. 365-92 or in Imai-Nishiya etal., (2007), BMC Biotechnol. 7: 84.

Also provided by the present invention is an antibody or fragmentthereof that binds to TL1A and which binds to the same epitope as theantibody comprising the heavy chain variable sequence comprising theamino acid sequence of SEQ ID NO. 27 or 29 and the light chain variablesequence comprising the amino acid sequence of SEQ ID NO. 14. Alsoprovided by the present invention is a specific region or epitope ofTL1A, which is bound by an antibody provided by the present invention,in particular by an antibody comprising the heavy chain variablesequence comprising the amino acid sequence of SEQ ID NO. 27 or SEQ IDNO 29 and the light chain variable sequence comprising the amino acidsequence of SEQ ID NO. 14. This specific region or epitope of the TL1Apolypeptide can be identified by any suitable epitope mapping methodknown in the art in combination with any one of the antibodies providedby the present invention. Examples of such methods include screeningpeptides of varying lengths derived from TL1A for binding to theantibody of the present invention with the smallest fragment that canspecifically bind to the antibody containing the sequence of the epitoperecognised by the antibody. The TL1A peptides may be producedsynthetically or by proteolytic digestion of the TL1A polypeptide.Peptides that bind the antibody can be identified by, for example, massspectrometric analysis. In another example, NMR spectroscopy or X-raycrystallography can be used to identify the epitope bound by an antibodyof the present invention. Once identified, the epitopic fragment whichbinds an antibody of the present invention can be used, if required, asan immunogen to obtain additional antibodies which bind the sameepitope.

Anti-TL1A Antibody Properties

Standard assays to evaluate the binding ability of the antibodies towarde.g. TL1A are known in the art, including for example, ELISAs, BIAcore®,Western blots, RIAs, and flow cytometry analysis. Suitable assays aredescribed in detail in the Examples. The binding kinetics (e.g., bindingaffinity like KD) of the antibodies also can be assessed by standardassays known in the art, such as by Scatchard or BIAcore® systemanalysis. The relative binding affinity K_(i) can be assessed bystandard competition assays known in the art.

In a further aspect the present invention provides antibodies orfragment thereof that bind to human, mouse, rat and cynomologus monkeyTL1A as visualized by ELISA or BIAcore® methods. Binding ELISA can becarried out and measured according to Example 3.

In a further aspect the present invention provides antibodies orfragments thereof that bind to recombinant or naturally produced humanTL1A and prevent activation and cytokine secretion by CD4 T lymphocytes.For example, the antibodies or fragments thereof of the invention maysuppress the production of INFγ induced by immune complex stimulatedmonocytes. An assay to determine such TL1A-mediated cytokine secretionby CD4 T lymphocytes can be carried out and measured according toExamples 3 and 6.

In a further aspect the present invention provides antibodies orfragment thereof that bind to TL1A, in particular TL1A in isolated form,with an affinity (K_(D)) of 850 pM or less, preferably 700 nM or less,more preferably 300 nM or less, more preferably 260 nM or less, evenmore preferably 250 nM or less, e.g. measured by Surface PlasmonResonance (SPR) on a BIAcore® instrument (GE Healthcare Europe GmbH,Glattbrugg, Switzerland) by capturing the antibody on a protein-Acoupled CM5 research grade sensor chip (GE Healthcare Europe GmbH,Glattbrugg, Switzerland; BR-1000-14) with a human soluble TL1Apolypeptide (encoded by SEQ ID NO: 116) used as analyte as detailed inExample 5. In a preferred aspect, the present invention provides ahumanized antibody or fragment thereof that retains at least 85% of theTL1A binding affinity (K_(D)) of the corresponding chimeric antibody.Preferably the humanized antibody or fragment thereof retains at least90% of the TL1A binding affinity (K_(D)) of the corresponding chimericantibody, more preferably at least 95% of the binding affinity (K_(D))of the corresponding chimeric antibody. Preferably, the humanizedantibody or fragment thereof binds human TL1A with equivalent affinityto the corresponding chimeric antibody. By “equivalent affinity” ismeant an affinity value that is within a range of ±10% of the TL1Abinding affinity of the corresponding chimeric antibody. Morepreferably, the present invention provides a humanized antibody orfragment thereof that binds human TL1A with a higher affinity than thecorresponding chimeric antibody. Preferably the humanized antibody orfragment thereof binds human TL1A with two-fold higher affinity than thecorresponding chimeric antibody, more preferably with three-fold higheraffinity than the corresponding chimeric antibody. In a preferred aspectof the present invention, humanized antibodies or fragment thereof thatbind to human TL1A are provided that have a binding affinity (K_(D)) of900 pM or less, 700 pM or less, preferably 500 pM or less, morepreferably 300 nM or less, more preferably 260 pM or less, even morepreferably 250 pM or less e.g. measured by Surface Plasmon Resonance(SPR) on a BIAcore® instrument (GE Healthcare Europe GmbH, Glattbrugg,Switzerland) by capturing the antibody on a protein-A coupled CM5research grade sensor chip (GE Healthcare Europe GmbH, Glattbrugg,Switzerland; BR-1000-14) with a human soluble TL1A polypeptide (encodedby SEQ ID NO: 116) used as analyte as detailed in Example 5.

A further aspect of the present invention provides antibodies orfragments thereof that bind to TL1A and which have good thermalstability. In a preferred embodiment, an antibody or fragment thereofthat binds to TL1A has a FAB fragment thermostability temperaturegreater than 70° C., preferably greater than 75° C. and even morepreferably greater than 80° C. For analysis of FAB fragmentthermostability differential scanning calorimetry measurements are used,whereas a mid-point melting temperature of the FAB fragment in contextof a full-length IgG is identified. These kind of calorimetricmeasurements are known to the skilled person and can be carried outaccording to e.g. Garber E & Demarest S J (2007) Biochem Biophys ResCommun, 355: 751-7, as further described in Example 5.

Nucleic Acids, Vectors and Host Cells

The present disclosure also provides isolated nucleic acids encoding theantibodies and fragments thereof that bind to TL1A, vectors and hostcells comprising the nucleic acid or the vector. The nucleic acids maybe present in whole cells, in a cell lysate, or in a partially purifiedor substantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art, see e.g. F. Ausubel, et al., ed. (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York. Anucleic acid of the invention can be, for example, DNA or RNA and may ormay not contain intron sequences. In a preferred embodiment, the nucleicacid is a cDNA molecule.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques e.g. cDNAs encoding the light and heavy chains of theantibody or encoding VH and VL segments can be obtained by standard PCRamplification or cDNA cloning techniques. For antibodies obtained froman immunoglobulin gene library (e.g., using phage display techniques),one or more nucleic acids encoding the antibody can be recovered fromthe library. The methods of introducing exogenous nucleic acid into hostcells are well known in the art, and will vary with the host cell used.Techniques include but are not limited to dextran-mediated transfection,calcium phosphate precipitation, calcium chloride treatment,polyethylenimine mediated transfection, polybrene mediated transfection,protoplast fusion, electroporation, viral or phage infection,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei. In the case of mammalian cells,transfection may be either transient or stable.

Preferred nucleic acids molecules of the invention are those encodingthe heavy chain sequence selected from the group consisting of SEQ IDNOS: 42, 43, 44, 45 and 46 and/or the light chain sequence selected fromthe group consisting of SEQ ID NOS: 47 and 48. Preferred nucleic acidsmolecules of the invention are those encoding the heavy chain variableregion selected from the group consisting of SEQ ID NOS: 31, 32, 33, 34and 35 and/or the light chain variable region selected from the groupconsisting of SEQ ID NOS: 36 and 37.

Preferred nucleic acids molecules of the invention are those encodingthe heavy chain variable region of SEQ ID NO: 1 and/or the light chainvariable region of SEQ ID NO: 2, e.g. DNA encoding the heavy chainvariable region comprising the nucleic acid sequence of SEQ ID NO: 63and/or DNA encoding the light chain variable region comprising thenucleic acid sequence of SEQ ID NO: 64. More preferred nucleic acidmolecules of the invention are those encoding the heavy chain variableregion of SEQ ID NOS: 27 or 29 and/or the light chain variable region ofSEQ ID NO: 14, e.g. DNA encoding the heavy chain variable regioncomprising the nucleic acid sequence of SEQ ID NOS: 33 or 35 and/or DNAencoding the light chain variable region comprising the nucleic acidsequence of SEQ ID NO: 36, which are most preferred.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, or to fragments genes corresponding tothe fragments described supra like Fab fragment genes or to a scFv gene.In these manipulations, a VL- or VH-encoding DNA fragment is operativelylinked to another DNA fragment encoding another protein, such as anantibody constant region or a flexible linker. The term “operativelylinked”, as used in this context, is intended to mean that the two DNAfragments are joined such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame. The isolated DNA encoding the VHregion can be converted to a full-length heavy chain gene by operativelylinking the VH-encoding DNA to another DNA molecule encoding heavy chainconstant regions (CH1, CH2 and CH3). The sequences of human heavy chainconstant region genes are known in the art (see e.g., Kabat E A et al.,supra) and DNA fragments encompassing these regions can be obtained bystandard PCR amplification. The heavy chain constant region can be anIgG1 (IGHG1), IgG2 (IGHG2), IgG3 (IGHG3), IgG4 (IGHG4), IgA1 (IGHA1),IgA2 (IGHA2), IgM (IGHM), IgD (IGHD), or IgE (IGHE) constant region, butmost preferably is an IgG1 (IGHG1) constant region. For a Fab fragmentheavy chain gene, the VH-encoding DNA can be operatively linked toanother DNA molecule encoding only the heavy chain CH1 constant region.The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat E A et al.,supra.) and DNA fragments encompassing these regions can be obtained bystandard PCR amplification. In preferred embodiments, the light chainconstant region can be a kappa or lambda constant region, preferably akappa constant region. To create a scFv gene, the VH- and VL-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3,such that the VH and VL sequences can be expressed as a contiguoussingle-chain protein, with the VL and VH regions joined by the flexiblelinker (see e.g., Bird R E et al., (1988) Science, 242: 423-426; HustonJ S et al., (1988) Proc. Natl. Acad. Sci. USA, 85: 5879-83; McCafferty Jet al., (1990) Nature, 348: 552-554). Various techniques have beendeveloped for the production of antibody fragments of antibodies.Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto K et al., (1992) J. Biochem. &Biophysical Methods, 24: 107-117 and Brennan M et al., (1985) Science,229: 81-3). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter P et al.,(1992) Bio/Technology, 10: 163-167). According to another approach,F(ab′)2 fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single-chain Fv fragment (scFv), see e.g. WO1993/16185; U.S. Pat. No. 5,571,894 and U.S. Pat. No. 5,587,458. Theantibody fragment may also be a “linear antibody”, e.g., as described inU.S. Pat. No. 5,641,870, for example.

The nucleic acids that encode the antibodies of the present inventionmay be incorporated into a vector, preferably an expression vector inorder to express the protein. A variety of expression vectors may beutilized for protein expression. Expression vectors may compriseself-replicating extra-chromosomal vectors or vectors which integrateinto a host genome. Expression vectors are constructed to be compatiblewith the host cell type. Thus vectors, preferably expression vectors,which find use in the present invention include but are not limited tothose which enable protein expression in mammalian cells, bacteria,insect cells, yeast, and in in vitro systems. As is known in the art, avariety of expression vectors are available, commercially or otherwise,that may find use in the present invention for expressing antibodies.

Expression vectors typically comprise a protein operably linked withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. By “operably linked” herein ismeant that the nucleic acid is placed into a functional relationshipwith another nucleic acid sequence. The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the antibody chain genes. Such regulatory sequencesare described, for example, in Goeddel (Gene Expression Technology,Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)).Generally, these expression vectors include transcriptional andtranslational regulatory nucleic acid operably linked to the nucleicacid encoding the antibody, and are typically appropriate to the hostcell used to express the protein. In general, the transcriptional andtranslational regulatory sequences may include promoter sequences,ribosomal binding sites, transcriptional start and stop sequences,translational start and stop sequences, and enhancer or activatorsequences. As is also known in the art, expression vectors typicallycontain a selection gene or marker to allow the selection of transformedhost cells containing the expression vector. Selection genes are wellknown in the art and will vary with the host cell used. For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes for this purpose include eubacteria, including gram-negativeor gram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.Suitable E. coli cloning hosts include E. coli 294 (ATCC 31,446), E.coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325). Inaddition to prokaryotes, eukaryotic microbes such as filamentous fungior yeast are suitable cloning or expression hosts. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species, and strains are commonly available and useful, such asSchizosaccharoriyces pombe; Kluyveromyces hosts including K. lactis, K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC24,178), K. WaItH (AJCC 56,500), K. drosopmarum (ATCC 36,906), K.thermotolerans, or K. marxianusyarrowia (EP402226); Pichia pastoris(EP183070); Candida; Trichoderma reesia (EP244234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi including Neurospora, Penicillium, Tolypocladium, or Aspergillushosts such as A. nidulans or A. niger.

Suitable host cells for the expression of the antibodies of theinvention are derived from multicellular organisms. Examples ofinvertebrate cells include plaril and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes augypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly) and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, for example, the L-1variant of Autographa californica NPV and the Bm-5 strain of Bombyx moriNPV, and such viruses may be used, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures of cotton, corn,potato, soybean, petunia, tomato, and tobacco can also be utilized ashosts.

Host cells for expressing the recombinant antibodies of the inventionare preferably mammalian host cells which include Chinese Hamster Ovary(CHO cells) (including dhfr⁻ CHO cells, described in Urlaub G & Chasin LA (1980) Proc. Natl. Acad. Sci, USA, 77: 4216-4220, used with a DHFRselectable marker, e.g., as described in Kaufman R J & Sharp P A (1982)J. Mol. Biol, 159: 601-621), NSO myeloma cells, COS cells and SP2 cells.In particular, for use with NSO myeloma cells, another preferredexpression system is the GS gene expression system disclosed in WO87/04462 (to Wilson), WO 89/01036 (to Bebbington) and EP338841 (toBebbington). When recombinant antibody genes are introduced intomammalian host cells, the antibodies are produced by culturing the hostcells for a period of time sufficient to allow for expression of theantibody in the host cells or, more preferably, for secretion of theantibody into the culture medium in which the host cells are grown. Hostcells useful for producing antibodies that bind to TL1A may be culturedin a variety of media. Commercially available media such as Ham's F10(Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), Minimal EssentialMedium (MEM; Sigma-Aldrich Chemie GmbH), RPMI-1640 (Sigma-Aldrich ChemieGmbH, Basel, Switzerland), and Dulbecco's Modified Eagle's Medium((DMEM; Sigma-Aldrich Chemie GmbH) are suitable for culturing the hostcells. Antibodies can be recovered from the culture medium usingstandard protein purification methods.

Antibodies may be operably linked to a fusion partner to enabletargeting of the expressed protein, purification, screening, display,and the like. Fusion partners may be linked to the antibody sequence viaa linker sequences. The linker sequence will generally comprise a smallnumber of amino acids, typically less than ten, although longer linkersmay also be used. Typically, linker sequences are selected to beflexible and resistant to degradation. As will be appreciated by thoseskilled in the art, any of a wide variety of sequences may be used aslinkers. For example, a common linker sequence comprises the amino acidsequence GGGGS. A fusion partner may be a targeting or signal sequencethat directs antibody and any associated fusion partners to a desiredcellular location or to the extracellular media. As is known in the art,certain signalling sequences may target a protein to be either secretedinto the growth media, or into the periplasmic space, located betweenthe inner and outer membrane of the cell. A fusion partner may also be asequence that encodes a peptide or protein that enables purificationand/or screening. Such fusion partners include but are not limited topolyhistidine tags (His-tags) (for example H6 and H10 or other tags foruse with Immobilized Metal Affinity Chromatography (LMAC) systems (e.g.Ni⁺² affinity columns)), GST fusions, MBP fusions, Strep-tag, the BSPbiotinylation target sequence of the bacterial enzyme BirA, and epitopetags which are targeted by antibodies (for example c-myc tags,flag-tags, and the like). As will be appreciated by those skilled in theart, such tags may be useful for purification, for screening, or both.

Construction and Production of Antibodies

Antibodies generated against the TL1A polypeptide may be obtained byimmunisation of an animal i.e. by administering the polypeptides to ananimal, preferably a non-human animal, using well-known and routineprotocols, see for example Handbook of Experimental Immunology (Weir D M(ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986).Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows,camels or pigs may be immunized. However, mice, rabbits, pigs and ratsin particular mice are generally most suitable. Antibodies can beproduced as well by recombinant DNA techniques known to the skilledperson. In additional antibodies can be produced by enzymatic orchemical cleavage of naturally occurring antibodies. Humanizedantibodies of the present invention may be constructed by transferringone or more CDRs or portions thereof from VH and/or VL regions from anon-human animal (e.g., mouse) to one or more framework regions fromhuman VH and/or VL regions. Optionally, human framework residues thuspresent in the VH and/or VL regions may be replaced by correspondingnon-human (e.g., mouse) residues when needed or desired for decreasingimmunogenicity of the antibody and/or maintaining binding affinity.Optionally, non-human amino acid residues present in the CDRs may bereplaced with human residues. Chimeric or humanized antibodies of thepresent invention can be prepared based on the sequence of a non-humanmonoclonal antibody prepared as described above. DNA encoding the heavyand light chain immunoglobulins can be obtained from the non-humanhybridoma of interest and engineered to contain non-murine (e.g., human)immunoglobulin sequences using standard molecular biology techniques.For example, to create a chimeric antibody, murine variable regions canbe linked to human constant regions using methods known in the art (seee.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). To create a humanizedantibody, murine CDR regions can be inserted into a human frameworkusing methods known in the art (see e.g., U.S. Pat. No. 5,225,539 toWinter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370to Queen et al.).

Humanized antibodies of the present invention may be constructed whereinthe human acceptor molecule for the heavy chain variable region isselected based on homology considerations between potential acceptormolecule variable regions and the heavy chain variable region of themurine antibody. Gell dine candidate human acceptor molecules arepreferred to reduce potential immunogenicity. Germline databases aremade up of antibody sequences that read through the end of the heavychain FW3 region and partially into the CDR3 sequence. For selection ofa FW4 region, databases of mature antibody sequences which have beenderived from the selected germline molecule can be searched or antibodysequences which have been derived from the selected germline moleculefrom a human donor can be used. Human acceptor molecules are preferablyselected from the same heavy chain class as the murine donor molecule,and of the same canonical structural class of the variable region of themurine donor molecule. Secondary considerations for selection of thehuman acceptor molecule for the heavy chain variable region eludehomology in CDR length between the murine donor molecule and the humanacceptor molecule. Human acceptor antibody molecules are preferablyselected by homology search to the V-BASE database, although otherdatabases such as the Kabat and the public NCBI databases may be used aswell.

Humanized antibodies of the present invention may be constructed whereinthe human acceptor molecule for the light chain variable region isselected based on homology considerations between potential acceptormolecule variable regions and with the light chain variable region ofthe murine antibody. Germline candidate human acceptor molecules arepreferred to reduce potential immunogenicity. Germline databases aremade up of antibody sequences that read through the end of the heavychain FW3 region and partially into the CDR3 sequence. For selection ofa FW4 region, databases of mature antibody sequences which have beenderived from the selected germline molecule can be searched or antibodysequences which have been derived from the selected germline moleculefrom a human donor can be used. Human acceptor molecules are preferablyselected from the same light chain class as the murine donor molecule,and of the same canonical structural class of the variable region of themurine donor molecule. Secondary considerations for selection of thehuman acceptor molecule for the light chain variable region includehomology in CDR length between the murine donor molecule and the humanacceptor molecule. Human acceptor antibody molecules are preferablyselected by homology searches to the V-BASE database, and otherdatabases such as the Kabat and the public NCBI databases may be used aswell. Methods for humanizing a non-human antibody are described herein,including in Example 5, below.

The present invention provides a method of producing an antibody orfragment thereof that binds to TL1A comprising culturing a host cellcomprising an isolated nucleic acid encoding the antibody or fragmentthereof that binds to TL1A or a vector comprising an isolated nucleicacid encoding the antibody or fragment thereof that binds to TL1A sothat the nucleic acid is expressed and the antibody produced. Preferablythe antibody is isolated. For host cells, nucleic acids and vectors, theones described above can be used. Expression of the nucleic acids can beobtained by, e.g. a combination of recombinant DNA techniques and genetransfection methods as is well known in the art (e.g., Morrison S(1985) Science 229: 1202) and as further outlined above. For example, toexpress the antibodies, or antibody fragments thereof, DNAs encodingpartial or full-length light and heavy chains, can be obtained bystandard molecular biology techniques (e.g., PCR amplification or cDNAcloning using a hybridoma that expresses the antibody of interest) andthe DNAs can be inserted into vectors such as expression vectors. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the VH segmentis operatively linked to the CH1 segment(s) within the vector and the VKsegment is operatively linked to the CK segment within the vector.

Characterization and Purification of Anti-TL1A Antibodies

Screening for antibodies can be performed using assays to measurebinding to TL1A and/or assays to measure the ability to block thebinding of TL1A to its receptor TNFRSF25. An example of a binding assayis an ELISA, in particular, using a fusion protein of TL1A and human Fc,which is immobilized on plates, and employing a conjugated secondaryantibody to detect anti-TL1A antibody bound to the fusion protein. Anexample of a blocking assay is a flow cytometry based assay measuringthe blocking of TL1A fusion protein binding to TNFRSF25 on human CD4cells. A fluorescently labelled secondary antibody is used to detect theamount of TL1A fusion protein binding to the cell. This assay is lookingfor a reduction in signal as the antibody in the supernatant blocks thebinding of ligand fusion protein to TNFRSF25. A further example of ablocking assay is an assay where the blocking of costimulation of naivehuman T cells mediated by TL1A fusion protein coated to a plate ismeasured by measuring thymidine incorporation. As an assay forevaluating the functional activity of anti-TL1A antibodies e.g. thereduction of cytokine secretion by CD4 T lymphocytes as described inExamples 3 and 6 can be used.

Antibodies of the present invention may be isolated or purified in avariety of ways known to those skilled in the art. Standard purificationmethods include chromatographic techniques, including ion exchange,hydrophobic interaction, affinity, sizing or gel filtration, andreversed-phase, carried out at atmospheric pressure or at high pressureusing systems such as FPLC and HPLC. Purification methods also includeelectrophoretic, immunological, precipitation, dialysis, andchromatofocusing techniques. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.To purify TL1A antibodies, selected host cells can be grown in e.g.spinner-flasks for monoclonal antibody purification. Supernatants can befiltered and concentrated before affinity chromatography with proteinA-sepharose (Pharmacia, Piscataway, N.J.). Eluted antibodies can bechecked by gel electrophoresis and high performance liquidchromatography to ensure purity. A preferred antibody of the presentinvention is thus an isolated and/or purified antibody that binds toTL1A.

Immunoconjugates

In another aspect, the present invention provides a TL1A antibody or afragment thereof that binds to TL1A, linked to a therapeutic agent, suchas a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.Such conjugates are referred to herein as “immunoconjugates”.Immunoconjugates that include one or more cytotoxins are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). Other examples of therapeuticcytotoxins that can be linked to an antibody of the invention includeduocarmycins, calicheamicins, maytansines and auristatins, andderivatives thereof. An example of a calicheamicin antibody conjugate iscommercially available (Mylotarg(R); American Home Products). Cytotoxinscan be linked to antibodies of the invention using linker technologyavailable in the art. Examples of linker types that have been used toconjugate a cytotoxin to an antibody include, but are not limited to,hydrazones, thioethers, esters, disulfides and peptide-containinglinkers. A linker can be chosen that is, for example, susceptible tocleavage by low pH within the lysosomal compartment or susceptible tocleavage by proteases, such as proteases preferentially expressed intumor tissue such as cathepsins (e.g., cathepsins B, C, D). For furtherdiscussion of types of cytotoxins, linkers and methods for conjugatingtherapeutic agents to antibodies, see also Saito G et al., (2003) Adv.Drug Deliv. Rev. 55: 199-215; Trail P A et al., (2003) Cancer Immunol.Immunother. 52: 328-337; Payne G (2003) Cancer Cell, 3: 207-212; AllenTM (2002) Nat. Rev. Cancer, 2: 750-763; Pastan I & Kreitman R J (2002)Curr. Opin. Investig. Drugs, 3: 1089-1091; Senter P D & Springer C J,(2001) Adv. Drug Deliv. Rev. 53: 247-264. Antibodies of the presentinvention also can be linked to a radioactive isotope to generatecytotoxic radiopharmaceuticals, also referred to asradioimmunoconjugates. Examples of radioactive isotopes that can beconjugated to antibodies for use diagnostically or therapeuticallyinclude, but are not limited to, iodine¹³¹, indium¹¹¹, yttrium⁹⁰ andlutetium¹⁷⁷. Methods for preparing radio-immunconjugates are establishedin the art. Examples of radioimmunoconjugates are commerciallyavailable, including Zevalin® (EDEC Pharmaceuticals) and Bexxar® (CorixaPharmaceuticals) and similar methods can be used to prepareradioimmunoconjugates using the antibodies of the invention. Theantibody immunoconjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (IL-1), interleukin-2(IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or othergrowth factors.

Techniques for linking such therapeutic agents to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al., (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al., (eds.), pp.475-506 (1985); “Analysis, Results, and Future Prospective of theTherapeutic Use of Radiolabeled Antibody in Cancer Therapy”, inMonoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al.,(eds.), pp. 303-16 (Academic Press 1985), and Thorpe P E & Ross W C(1982) Immunol. Rev. 62: 119-58.

In another aspect, the present invention provides a TL1A antibody or afragment thereof that binds to TL1A, administered together with atherapeutic agent, such as a cytotoxin, a drug (e.g., animmunosuppressant) or a radiotoxin.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, comprising the antibody or fragment thereof,of the present invention, and a pharmaceutically acceptable carrier.Such compositions may include one or a combination of (e.g., two or moredifferent) antibodies, and/or immunoconjugates of the invention and/or atherapeutic agent, such as a cytotoxin, a drug (e.g., animmunosuppressant) or a radiotoxin as described supra. For example, apharmaceutical composition of the invention can comprise a combinationof antibodies (or immunoconjugates) that bind to different epitopes onthe target antigen or that have complementary activities. Pharmaceuticalcompositions of the invention also can be administered in combinationtherapy, i.e., combined with other agents. For example, the combinationtherapy can include a TL1A antibody of the present invention combinedwith at least one other anti-inflammatory or immunosuppressant agent.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody orimmunoconjugate, may be coated in a material to protect the compoundfrom the action of acids and other natural conditions that mayinactivate the compound. Pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

In another aspect, the present invention provides a compositioncomprising an immunoconjugate comprising the antibody or fragmentthereof that binds to TL1A linked to a therapeutic agent and apharmaceutically acceptable carrier. Immunoconjugates and therapeuticagents which can be used are as described supra.

In another aspect, the present invention provides a compositioncomprising the antibody or fragment thereof of the present inventionwhich further comprises another pharmaceutically active agent.Preferably the another pharmaceutically active agent is one or more of:a) another antagonist to TL1A, b) an anti-inflammatory agent, c) animmune suppressive agent e.g. TNFα antagonist, cortisone or steroidsetc) and/or d) an anti-allergy agent.

A pharmaceutical composition of the invention may also include apharmaceutically acceptable antioxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic-acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like. Examples of suitable aqueous andnonaqueous carriers that may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. These compositions mayalso contain adjuvants such as preservatives, wetting agents,emulsifying agents and dispersing agents. Prevention of presence ofmicroorganisms may be ensured both by sterilization procedures, supra,and by the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

Therapeutic and Other Uses

The antibodies of the present invention have numerous in vitro and invivo diagnostic and therapeutic utilities involving the diagnosis andtreatment of TL1A mediated disorders. For example, these molecules canbe administered to cells in culture, in vitro or ex vivo, or to humansubjects, e.g., in vivo, to treat, prevent and to diagnose a variety ofTL1A-mediated disorders. Preferred subjects are human and includepatients having disorders mediated by TL1A activity (TL1A mediateddisorders). The neutralizing antibodies of the present invention can beeffective in treating patients independent of their TL1A costimulatorystatus. More preferred subjects are human and include patientsexpressing a high level of TL1A.

A “patient” for the purposes of the present invention includes bothhumans and other animals, preferably mammals and most preferably humans.Thus the antibodies of the present invention have both human therapy andveterinary applications. The term “treatment” or “treating” in thepresent invention is meant to include therapeutic treatment, as well asprophylactic, or suppressive measures for a disease or disorder. Thus,for example, successful administration of an antibody prior to onset ofthe disease results in treatment of the disease. As another example,successful administration of an antibody after clinical manifestation ofthe disease to combat the symptoms of the disease comprises treatment ofthe disease. “Treatment” and “treating” also encompasses administrationof an antibody after the appearance of the disease in order to eradicatethe disease. Successful administration of an antibody after onset andafter clinical symptoms have developed, with possible abatement ofclinical symptoms and perhaps amelioration of the disease, comprisestreatment of the disease. Those “in need of treatment” include mammalsalready having the disease or disorder, as well as those prone to havingthe disease or disorder, including those in which the disease ordisorder is to be prevented.

In a particular embodiment, the antibodies are used in vivo to treat,prevent or diagnose a variety of TL1A-mediated disorders. Thus theinvention provides a method for treating a TL1A mediated disorder in asubject, the method comprising administering to the subject atherapeutically effective amount of the antibody or fragment thereof.Exemplary TL1A mediated disorders include, but are not limited to,inflammatory diseases and/or autoimmune diseases, for example,inflammatory bowel disease (IBD) including ulcerative colitis andCrohn's disease, rheumatoid arthritis, MS, type 1 and type 2 diabetes,psoriasis, psoriatic arthritis, ankylosing spondylitis, atopicdermatitis; allergic reactions or conditions, including for example,asthma and allergic lung inflammation; cancers, atherosclerosis,infections, neurodegenerative diseases, graft rejection, graft versushost diseases (GVHD) and cardiovascular disorders/diseases. Theinvention also provides a method for treating a TL1A mediated disorderin a subject, the method comprising administering to the subject atherapeutically effective amount of the antibody or fragment thereof.Exemplary TL1A mediated disorders include, but are not limited to,inflammatory diseases and/or autoimmune diseases, for example,inflammatory bowel disease (IBD) including ulcerative colitis andCrohn's disease, rheumatoid arthritis, MS, type 1 and type 2 diabetes,psoriasis, psoriatic arthritis, ankylosing spondylitis, atopicdermatitis; allergic reactions or conditions, including for example,asthma and allergic lung inflammation; cancers, atherosclerosis,infections, neurodegenerative diseases, graft rejection, graft versushost diseases (GVHD) and cardiovascular disorders/diseases, chronicobstructive pulmonary disease COPD, optic neuritis, age related maculardegeneration, systemic lupus erythematosus (SLE), sjogen's syndrome,scleroderma, systemic sclerosis, chronic Kidney disease, liver fibrosis,tuberculosis, idiopathic pulmonary fibrosis, tuberculosis induced lungfibrosis, retroperitoneal Fibrosis, pulmonary fibrosis, cystic fibrosis,endomyocardial fibrosis, atrial fibrosis, mediastinal fibrosis,myelofibrosis (bone marrow), retroperitoneal fibrosis, progressivemassive fibrosis, pephrogenic systemic fibrosis, arthrofibrosis.Preferably, the TL1A mediated disorders include inflammatory diseasesand/or auto immune diseases, including inter alia inflammatory boweldiseases (e.g., ulcerative colitis and Crohn's disease), rheumatoidarthritis, MS and atherosclerosis.

Preferred TL1A mediated disorders to be treated with the antibody of theinvention are selected from the group consisting of inflammatory boweldisease, multiple sclerosis, rheumatoid arthritis and asthma. Aparticular preferred TL1A mediated disorder to be treated with theantibody of the invention is inflammatory bowel disease.

Animal model for evaluating the functional activity of anti-TL1Aantibodies in TL1A-mediated disorders are described in Example 7 forasthma and in Examples 8 and 9 for IBD.

In one embodiment, the antibodies of the invention can be used to detectlevels of TL1A, or levels of cells which contain TL1A on their membranesurface, which levels can then be linked to certain disease symptoms.Alternatively, the antibodies can be used to inhibit or block TL1Afunction which, in turn, can be linked to the prevention or ameliorationof certain disease symptoms, thereby implicating TL1A as a mediator ofthe disease. This can be achieved by contacting a sample and a controlsample with the TL1 antibody under conditions that allow for theformation of a complex between the antibody and TL1A. Any complexesformed between the antibody and TL1A are detected and compared in thesample and the control. In light of the specific binding of theantibodies of the invention for TL1A, the antibodies of the inventioncan be used to specifically detect TL1A expression on the surface ofcells e.g. can be used to detect a patient having low or high expressionlevels of TL1A. The antibodies of the invention can also be used topurify TL1A via immunoaffinity purification.

In another embodiment, the antibodies of the invention can be initiallytested for binding activity associated with therapeutic or diagnosticuse in vitro. For example, compositions of the invention can be testedusing flow cytometric assays.

The present disclosure further provides the use of an antibody orfragment thereof as a medicament and the use of an antibody or fragmentthereof in the preparation of a medicament for the treatment of a TL1Amediated disorder. In a further embodiment the present disclosureprovides the antibody or fragment thereof for use as a medicament. Alsoprovided by the present disclosure is the antibody or fragment thereoffor use in a method for treating a TL1A mediated disorder. TL1A mediateddisorders are the ones as described supra. The antibody or fragmentthereof of the present invention may be particularly useful for treatingTL1A mediated disorders independent of the DR3 costimulatory status of apatient. In a preferred embodiment, the antibody or fragment thereof canbe used for treating a TL1A mediated disorder wherein a patientexpresses a high level of TL1A.

As previously described, anti-TL1A antibodies of the invention can beco-administered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immunoconjugate as describedsupra) or can be administered separate from the agent. In the lattercase (separate administration), the antibody can be administered before,after or concurrently with the agent or can be co-administered withother known therapies, e.g., an anti-cancer therapy, e.g., radiation.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 10 mg/kg, of the host bodyweight. An exemplary treatment regime entails administration once perweek, once every two weeks, once every three weeks, once every fourweeks, once a month, once every three months or once every three to sixmonths. The antibody is usually administered on multiple occasions.Intervals between single dosages can be, for example, weekly, monthly,every three months or yearly. Intervals can also be irregular asindicated by measuring blood levels of antibody to the target antigen inthe patient. In some methods, dosage is adjusted to achieve a plasmaantibody concentration of about 1-1000 μg/ml and in some methods about25-300 μg/ml. Alternatively the antibody can be administered as asustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the antibody in the patient. The dosage and frequency ofadministration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated.

Actual dosage levels of the active ingredients, i.e. the antibody in thepharmaceutical compositions of the present invention may be varied so asto obtain an amount of the active ingredient which is effective toachieve the desired therapeutic response for a particular patient,composition, and mode of administration, without being toxic to thepatient. The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions of the present invention employed, the route ofadministration, the time of administration, the rate of excretion of theparticular antibody being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective amount” of a TL1A antibody of the inventionpreferably results in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods,and/or a prevention of impairment or disability due to the diseaseaffliction. The ability of a compound for the treatment of a TL1Amediated disorder can be evaluated in an animal model system predictiveof efficacy in human. Alternatively, this property of a composition canbe evaluated by examining the ability of the compound to inhibit cellgrowth, such inhibition can be measured in vitro by assays known to theskilled practitioner. One of ordinary skill in the art would be able todetermine such amounts based on such factors as the subject's size, theseverity of the subject's symptoms, and the particular composition orroute of administration selected.

The antibody or the composition of the present invention can beadministered via one or more routes of administration using one or moreof a variety of methods known in the art. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results. Preferred routes of administrationinclude intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. More preferred routes ofadministration are intravenous or subcutaneous. The phrase “parenteraladministration” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. Alternatively, an antibody of theinvention can be administered via a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually or topically.

Article of Manufacture and Kit

In another embodiment of the disclosure, an article of manufacturecomprising the antibody or fragment thereof, the composition or theimmunoconjugate of the invention for the treatment of a TL1A mediateddisorder is provided. The article of manufacture may comprise acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials orsyringes. The containers may be formed from a variety of materials suchas glass or plastic. The container holds a composition that may beeffective for treating the condition and may have a sterile access port(e.g., the container may be an intravenous solution bag or a vial havinga stopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition may be the antibody described herein.The label or package insert may indicate that the composition may beused for treating the condition of choice, such as cancer. In oneembodiment, the label or package insert may indicate that thecomposition comprising the antibody may be used to treat a TL1A-mediateddisorder.

Moreover, the article of manufacture may comprise (a) a first containerwith a composition contained therein, wherein the composition comprisesthe antibody herein, and (b) a second container with a compositioncontained therein, wherein the composition comprises a therapeutic agentother than the antibody. The article of manufacture in this embodimentof the disclosure may further comprise a package insert indicating thatthe first and second compositions can be used in combination to treat aTL1A mediated disease or disorder. Such therapeutic agent may be any ofthe adjunct therapies described in the preceding section (e.g., athrombolytic agent, an anti-platelet agent, a chemotherapeutic agent, ananti-angiogenic agent, an anti-hormonal compound, a cardioprotectant,and/or a regulator of immune function in a mammal, including acytokine). Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

Also within the scope of the present invention are kits comprising theantibody, the compositions or the immunoconjugates of the invention andinstructions for use. The kit can further contain one more additionalreagents, such as an immunosuppressive reagent, a cytotoxic agent or aradiotoxic agent, or one or more additional antibodies of the invention(e.g., an antibody having a complementary activity which binds to anepitope in the TL1A antigen distinct from the first antibody).

Without further description, it is believed that one of ordinary skillin the art may, using the preceding description and the followingillustrative examples, make and utilize the agents of the presentdisclosure and practice the claimed methods. The following workingexamples are provided to facilitate the practice of the presentdisclosure, and are not to be construed as limiting in any way theremainder of the disclosure.

EXAMPLES Example 1 Generation and Screening of Mouse Anti-Human TL1AAntibodies

To produce the recombinant human TL1A-Fc protein, a cDNA for the humanTL1A gene was purchased from Source BioScience (Nottingham, UK; clonenumber: IRATp970G02115D). This cDNA was used as a template to amplifythe coding region of the processed secreted version of human TL1A (SEQID NO: 116) using PCR. The PCR was performed using primers GlnPr994 andGlnPr995 (SEQ ID NOs: 119 and 120, respectively). Primer GlnPr994 adds aBamHI restriction site 5′ of the extracellular region and cleaves thenative signal peptide. Primer GlnPr995 adds a HindIII restriction site3′ of the extracellular region. The amplicon was cut using the flankingrestriction sites BamHI and HindIII and cloned into a modified mammalianexpression vector based on the pcDNA3.1(−) plasmid from Invitrogen(Invitrogen AG, Basel, Switzerland), expressing an Fc-fusion construct.The expression vector contains the human CMV promoter with the Ig donoracceptor fragment (first intron) described in U.S. Pat. No. 5,924,939,the OriP sequence (Koons M D et al., (2001) J Virol. 75(22): 10582-92),the SV40 enhancer, and the SV40 polyA fused to the gastrin terminator asdescribed by Kim D, et al., (2003) Biotechnol. Prog. 19(5): 1620-2. Theexpression cassette contains a kozak region upstream of the open readingframe of the Fc fusion protein, followed by a signal peptide, terminatedby a BamHI restriction site for convenient cloning. The Fc region of thefusion protein is started by a HindIII restriction site for convenientcloning, followed by a small glycine-serine linker. In order to releasethe previous construct upstream of the Fc region, the vector was cutusing BamHI (NEB, Ipswich, Mass., USA) and HindIII (NEB, Ipswich, Mass.,USA), treated using CIP (NEB, Ipswich, Mass., USA) and gel purified. Theinsert coding for the extracellular region of human TL1A was ligated inthe backbone and transformed in E.coli Top 10 cells (Life Technologies,Carlsbad, Calif., USA) leading to the Fc fusion expression construct(SEQ ID NO: 117). The first 20 amino acids of SEQ ID NO: 117 correspondto the signal sequence, which was not present in the final Fc fusionexpression construct.

This recombinant plasmid allowed for expression of the human TL1A-Fcfusion protein in mammalian cells with secretion into the cell culturemedium driven by the signal peptide. For recombinant protein production,the aforementioned recombinant vector was transfected intosuspension-adapted HEK 293 EBNA cells (Life Technologies, Carlsbad,Calif., USA) using jetPEI™ transfection reagent (Polyplus-transfectionS. A., Strasbourg, France; Distributor: Brunschwig, Basel, Switzerland).The cell culture supernatant was collected after five days and furtherpurified using a Protein A affinity purification column (HiTrap ProteinA sepharose column; GE Healthcare Europe GmbH, Glattbrugg, Switzerland)operated on an ÄKTA FPLC system (GE Healthcare Europe GmbH, Glattbrugg,Switzerland).

To produce the secreted recombinant human TL1A protein with his-tag, theDNA sequence coding for the processed version of secreted human TL1A wasamplified by PCR using primers GlnPr1542 and GlnPr1543 (SEQ ID NOs: 121and 122, respectively) adding an N-terminal 6× His linker. A secondround of PCR using primers GlnPr1544 (SEQ ID NO: 123) and GlnPR1543 addsa signal peptide and convenient flanking restriction sites for cloning(5′: NheI; 3′: XhoI). The PCR product was cut using NheI and XhoI (NEB,Ipswich, Mass., USA) and subsequently cloned in the modified pcDNA3.1(−)plasmid described above, cut using the same enzymes and CIPed. Thisrestriction digest releases the open reading frame of the entireFc-fusion protein previously present in the expression vector. Afterligation and transformation in Top 10 E. coli cells, the final plasmidwas chosen based on restriction digest and sequencing of the expressionconstruct for secreted TL1A with an N-terminal his-tag (SEQ ID No: 118).The first 20 amino acids of SEQ ID NO: 118 correspond to the signalsequence, which was not present in the final N-terminal his-tagexpression construct.

This recombinant plasmid allowed for the expression of the humanTL1A-his protein in mammalian cells with secretion into the cell culturemedia. For protein production, the recombinant vector was transfectedinto suspension-adapted HEK 293 EBNA cells (Life Technologies, Carlsbad,Calif., USA) using jetPEI™ transfection reagent (Polyplus-transfectionS. A., Strasbourg, France; Distributor: Brunschwig, Basel, Switzerland).The cell culture supernatant was collected five days after transfectionand purified using a Ni²⁺-NTA affinity purification column (HiTrapNi²⁺-NTA sepharose column; GE Healthcare Europe GmbH, Glattbrugg,Switzerland) operated on an AKTA FPLC system (GE Healthcare Europe GmbH,Glattbrugg, Switzerland). The buffer of the recombinant human TL1A-Fcand TL1A-his proteins was changed into phosphate buffer saline (PBS).Recombinant human TL1A-Fc protein dissolved in PBS was mixed with anequal volume of Stimune adjuvant (Prionics, Switzerland) and an emulsionwas prepared. The emulsion was transferred to 0.5 mL insulin syringes(BD Pharmingen, Allschwil, Switzerland) and BALB/c animals (Harlan,Netherlands) were immunized sub-cutaneously in the back footpads, thebase of the tail and the neck with 50 μg of the emulsified protein. Theimmunization was repeated two weeks later with the same amount ofantigen and the same route of injection.

The presence of circulating anti-human TL1A antibodies in the immunizedmouse sera was evaluated by direct ELISA using plates coated with therecombinant human TL1A-his protein. A serial dilution (from 1:10⁰ to1:10⁹) of the different mouse sera was added to the plates and the boundantibodies were detected using a goat anti-mouse H+L whole molecule-HRP(Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). A final sub-cutaneousboost with 50 μg of antigen without adjuvant was performed in animalsdisplaying the best anti-human TL1A IgG serum titer three days beforesacrifice.

Animals were euthanized and the inguinal, axillary, brachial, poplitealand sciatic lymph nodes were collected to prepare a single cellsuspension by disturbing the lymph node architecture with two 25Gneedles in a DNAse (Roche Diagnostics (Schweiz) AG, Rotkreuz,Switzerland) and collagenase (Roche Diagnostics (Schweiz) AG, Rotkreuz,Switzerland) solution. Single cell suspensions were fused to a myelomacell line X63AG8.653 (mouse BALB/c myeloma cell line; ATCC accessionnumber: CRL 1580; Kearney J F et al., (1979) J. Immunol. 123(4):1548-1550) at a ratio of 7:1 (fusion partner-to-harvested lymph nodecells) with polyethylene glycol 1500 (Roche Diagnostics (Schweiz) AG,Rotkreuz, Switzerland). The fused cells were plated into 96 well flatbottom plates containing mouse macrophages in DMEM-10 medium (InvitrogenAG, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS,PAA Laboratories, Pasching, Austria), 2 mM L-glutamine, 100 U/ml(Biochrom AG, Germany) penicillin, 100 ug/ml streptomycin (Biochrom AG,Germany), 10 mM HEPES (Invitrogen AG, Basel, Switzerland), 50 μMβ-mercaptoethanol (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), HAT(Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) and 1% Growth factor(Hybridokine, Interchini/Uptima, Montluçon, France).

Approximately 800 wells from the fusions were screened by ELISA for thepresence of mouse IgG that recognized human TL1A and blocked the bindingof human TL1A to its receptor. Positive wells were expanded andsubjected to two rounds of sub cloning. Cells were collected and theheavy and light chains were cloned and sequenced.

Example 2

Cloning and Sequencing of the VH and VL Chains of the Anti-TL1AAntibodies from Hybridoma Cells

For each positively selected hybridoma, total RNA was prepared,reverse-transcribed into cDNA and VH and VL genes were respectivelyamplified by PCR. These PCR products were ligated into a rescue-vector(pDrive vector; QIAGEN AG, Hombrechtikon, Switzerland), allowing for theDNA sequencing of individual PCR products and the determination of mono-or poly-clonality of the selected hybridomas. This vector allowed forblue/white selection on LB-agar plates containing IPTG and X-gal(colonies with no insert were blue because of the degradation of X-galby the LacZ α-peptide). Recombinant plasmids from positive (white)bacterial clones were prepared and sequenced using standard DNAsequencing primers specific for the vector backbone (M13rev, M13fwd, T7or SP6). DNA sequences were finally subcloned into an expression vectorfor recombinant expression of the antibody of interest in mammaliancells.

RNA Isolation

Total RNA was isolated from 2-10×10⁶ cells using the RNeasy Mini Kitfrom QIAGEN (QIAGEN AG, Hombrechtikon, Switzerland) according to themanufacturer's protocol; samples were quantified using a NanoDropND-1000 spectrophotometer (WITEC AG, Littau, Switzerland).

One Step RT-PCR

The total RNA preparations described above were furtherreverse-transcribed into cDNA, and the VH and VL fragments wereamplified by PCR using two different mixtures of degenerated primers,each one allowing the recovery of all the different subfamilies of mouseimmunoglobulin heavy chain variable fragments and variable heavy chainjunction regions or the recovery of all mouse immunoglobulin light chainkappa variable fragments and variable light chain kappa junctionregions. The primers used for reverse transcription and amplificationwere synthesized by Microsynth (Balgach, Switzerland), and were HPLCpurified (Tables 1-4). Both reverse-transcription and PCR amplificationwere performed simultaneously using the QIAGEN one step RT-PCR kit(QIAGEN AG, Hombrechtikon, Switzerland). Since the technique usedspecific primers, each mRNA sample was then treated in duplicateallowing for the individual reverse-transcription and amplification ofeither the VH or the VL fragments. 2 μg of total RNA dissolved intoRNase-free water to a final volume of 30 μg were mixed with: 10 μl of a5× stock solution of QIAGEN OneStep RT-PCR Buffer, 2 μl of a dNTPs mixat a concentration of 10 mM, 3 μl of primer mix at a concentration of 10μM and 2 μl of QIAGEN OneStep RT-PCR Enzyme Mix. The final mixture wasthen placed in a PCR tube, and cycled in a PCR-themocycler (BioRadiCycler version 4.006, Bio-Rad Laboratories AG, Reinach, Switzerland)using the following settings:

-   -   30 min at 50° C.    -   15 min at 95° C.

40 cycles: 30 sec at 94° C.

-   -   30 sec at 55° C.    -   1 min at 72° C.

10 min at 72° C.

-   -   Hold at 4° C.        pDrive Cloning

PCR products were run onto 2% agarose gels. Following DNAelectrophoresis, the fragments of interest (˜450 bp) were excised fromthe agarose gels, and further extracted using the Macherey-NagelNucloSpin Extract II kit 250 (Macherey-Nagel, Oensingen, Switzerland).For DNA sequencing, the extracted PCR products were cloned into therescue-vector described above (pDrive vector, QIAGEN AG, Hombrechtikon,Switzerland) and transformed into the E. coli TOP10 strain (InvitrogenAG, Basel, Switzerland).

Miniprep Extraction

Positive colonies were cultured overnight at 37° C. (shaking 250 RPM) in1.5 ml of Luria Bertani (LB) medium supplemented with 100 μg/mlampicillin seeded in Macherey-Nagel Square-well Block plates(Macherey-Nagel, Oensingen, Switzerland). The next day DNA miniprepextractions were performed using the Nucleo Spin Multi-8 Plasmid kit(Macherey-Nagel, Oensingen, Switzerland).

Sequencing and Sequence Snalysis

Samples were sent for DNA sequencing to the DNA sequencing servicecompany Fasteris (Plan-les-Ouates, Switzerland). The standard primers:M13rev, M13fwd, T7, SP6 were used (Table 5). To analyse the DNAsequences, the Clone Manager 9 Professional Edition (Scientific &Educational Software, NC, USA) and the BioEdit Sequence Alignment Editor(Hall T A (1999) Nucl Acids Symp Ser 41: 95-98) were used.

Cloning of Expression Vector for Recombinant Chimeric AntibodyExpression

For recombinant expression in mammalian cells, the isolated murine VHand VL fragments were formatted as chimeric immunoglobulins usingassembly-based PCR methods. These chimeric antibodies consist of a heavychain where the murine heavy chain variable domain is fused to the humanIgG1 heavy chain constant domains (γ1, hinge, γ2, and γ3 regions) and alight chain where the murine light chain variable domain is fused to ahuman kappa constant domain (Cκ). PCR-assembled murine variable andhuman constant parts were subsequently cloned into a modified mammalianexpression vector based on the modified pcDNA3.1(−) vector fromInvitrogen mentioned in Example 1 with the difference that a humanimmunoglobulin light chain kappa leader peptide was employed to driveprotein secretion. For protein production of the immunoglobulincandidates, equal quantities of heavy and light chain vector DNA wereco-transfected into suspension-adapted HEK-293 (ATCC number: CRL-1573).The cell culture supernatant was collected after five days and purifiedusing a Protein A affinity purification column (HiTrap Protein Asepharose column) operated on an AKTA FPLC system (both from GEHealthcare Europe GmbH, Glattbrugg, Switzerland).

TABLE 1 primer Mix VH-back  1GTG ATC GCC ATG GCG TCG ACC GAK GTR MAG CTT CAG GAG TC SEQ ID NO: 65  2GTG ATC GCC ATG GCG TCG ACC GAG GTB CAG CTB CAG CAG TC SEQ ID NO: 66  3GTG ATC GCC ATG GCG TCG ACC CAG GTG CAG CTG AAG SAR TC SEQ ID NO: 67  4GTG ATC GCC ATG GCG TCG ACC GAG GTC CAR CTG CAA CAR TC SEQ ID NO: 68  5GTG ATC GCC ATG GCG TCG ACC CAG GTY CAG CTB CAG CAR TC SEQ ID NO: 69  6GTG ATC GCC ATG GCG TCG ACC CAG GTY CAR CTG CAG CAR TC SEQ ID NO: 70  7GTG ATC GCC ATG GCG TCG ACC CAG GTC CAC GTG AAG CAR TC SEQ ID NO: 71  8GTG ATC GCC ATG GCG TCG ACC GAG GTG AAS STG GTG GAR TC SEQ ID NO: 72  9GTG ATC GCC ATG GCG TCG ACC GAV GTG AWG STG GTG GAG TC SEQ ID NO: 73 10GTG ATC GCC ATG GCG TCG ACC GAG GTG CAG STG GTG GAR TC SEQ ID NO: 74 11GTG ATC GCC ATG GCG TCG ACC GAK GTG CAM CTG GTG GAR TC SEQ ID NO: 75 12GTG ATC GCC ATG GCG TCG ACC GAG GTG AAG CTG ATG GAR TC SEQ ID NO: 76 13GTG ATC GCC ATG GCG TCG ACC GAG GTG CAR CTT GTT GAR TC SEQ ID NO: 77 14GTG ATC GCC ATG GCG TCG ACC GAR GTR AAG CTT CTC GAR TC SEQ ID NO: 78 15GTG ATC GCC ATG GCG TCG ACC GAA GTG AAR STT GAG GAR TC SEQ ID NO: 79 16GTG ATC GCC ATG GCG TCG ACC CAG GTT ACT CTR AAA SAR TC SEQ ID NO: 80 17GTG ATC GCC ATG GCG TCG ACC CAG GTC CAA CTV CAG CAR CC SEQ ID NO: 81 18GTG ATC GCC ATG GCG TCG ACC GAT GTG AAC TTG GAA SAR TC SEQ ID NO: 82 19GTG ATC GCC ATG GCG TCG ACC GAG GTG AAG GTC ATC GAR TC SEQ ID NO: 83

TABLE 2 primer Mix VH-forward 1CCTCCACCACTCGAGCC CGA GGA AAC GGT GAC CGT GGT SEQ ID NO: 84 2CCTCCACCACTCGAGCC CGA GGA GAC TGT GAG AGT GGT SEQ ID NO: 85 3CCTCCACCACTCGAGCC CGC AGA GAC AGT GAC CAG AGT SEQ ID NO: 86 4CCTCCACCACTCGAGCC CGA GGA GAC GGT GAC TGA GGT SEQ ID NO: 87

TABLE 3 primer Mix VL-back  1GGCGGTGGC GCT AGC GAY ATC CAG CTG ACT CAG CC SEQ ID NO: 88  2GGCGGTGGC GCT AGC CAA ATT GTT CTC ACC CAG TC SEQ ID NO: 89  3GGCGGTGGCGCT AGC GAY ATT GTG MTM ACT CAG TC SEQ ID NO: 90  4GGCGGTGGC GCT AGC GAY ATT GTG YTR ACA CAG TC SEQ ID NO: 91  5GGCGGTGGC GCT AGC GAY ATT GTR ATG ACM CAG TC SEQ ID NO: 92  6GGCGGTGGC GCT AGC GAY ATT MAG ATR AMC CAG TC SEQ ID NO: 93  7GGCGGTGGC GCT AGC GAY ATT CAG ATG AYD CAG TC SEQ ID NO: 94  8GGCGGTGGCGCT AGC GAY ATY CAG ATG ACA CAG AC SEQ ID NO: 95  9GGCGGTGGC GCT AGC GAY ATT GTT CTC AWC CAG TC SEQ ID NO: 96 10GGCGGTGGCGCT AGC GAY ATT GWG CTS ACC CAA TC SEQ ID NO: 97 11GGCGGTGGC GCT AGC GAY ATT STR ATG ACC CAR TC SEQ ID NO: 98 12GGCGGTGGC GCT AGC GAY RTT KTG ATG ACC CAR AC SEQ ID NO: 99 13GGCGGTGGCGCT AGC GAY ATT GTG ATG ACB CAG KC SEQ ID NO: 100 14GGCGGTGGC GCT AGC GAY ATT GTG ATA ACY CAG GA SEQ ID NO: 101 15GGCGGTGGC GCT AGC GAY ATT GTG ATG ACC CAG WT SEQ ID NO: 102 16GGCGGTGGC GCT AGC GAY ATT GTG ATG ACA CAA CC SEQ ID NO: 103 17GGCGGTGGCGCT AGC GAY ATT TTG CTG ACT CAG TC SEQ ID NO: 104 18GGCGGTGGC GCT AGC GAA ACA ACT GTG ACC CAG TC SEQ ID NO: 105 19GGCGGTGGCGCT AGC GAA AAT GTK CTS ACC CAG TC SEQ ID NO: 106 20GGCGGTGGCGCT AGC CAG GCT GTT GTG ACT CAG GAA TC SEQ ID NO: 107

TABLE 4 primer Mix VL-forward 1ATGCTGAC GC GGC CGC ACG TTT KAT TTC CAG CTT GG SEQ ID NO: 108 2ATGCTGAC GC GGC CGC ACG TTT TAT TTC CAA CTT TG SEQ ID NO: 109 3ATGCTGAC GC GGC CGC ACG TTT CAG CTC CAG CTT GG SEQ ID NO: 110 4ATGCTGAC GC GGC CGC ACC TAG GAC AGT CAG TTT GG SEQ ID NO: 111

TABLE 5 sequencing primers M13-Fwd GTAAAACGACGGCCAGT SEQ ID NO: 112M13-Rev AACAGCTATGACCATG SEQ ID NO: 113 T7 TAATACGACTCACTATAGGSEQ ID NO: 114 SP6 GATTTAGGTGACACTATAG SEQ ID NO: 115

Example 3 Biological Characterization of Anti-Human TL1A AntibodiesTL1A-Specific Antibody Detection ELISA

Antibody titers, specificity and production by hybridomas andrecombinant antibody candidates were determined by a direct ELISA.Briefly, 96 well-microtiter plates (Costar USA, distributor VWR AG,Nyon, Switzerland) were coated with 100 μl of recombinant human TL1A-hisat 2 μg/ml in PBS (see Example 1 for the generation of the TL1A-hisprotein). Plates were incubated overnight at 4° C. and were then blockedwith PBS 2% BSA (Bovine Serum Albumin, PAA Laboratories, Pasching,Austria) at room temperature (RT) for one hour. The blocking solutionwas removed and the hybridoma supernatants or purified antibodies wereadded. The plates were incubated at RT for 30 minutes, then washed ninetimes with PBS 0.01% Tween-20 (Sigma-Aldrich Chemie GmbH, Buchs,Switzerland) and a Horseradish Peroxidase (HRP) labelled-Goat anti-mouseH+L-detection antibody (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland)was added at a dilution of 1:1000. To detect recombinant chimericantibodies (see Example 2) that possess a human Fc, a HRP-labelledrabbit anti human IgG antibody (Sigma-Aldrich Chemie GmbH, Buchs,Switzerland) at a dilution of 1:1000 was used as the detection antibody.Plates were incubated for 30 minutes at room temperature (RT), washednine times with PBS 0.01% Tween-20 and the TMB substrate (Bio-radLaboratories AG, Reinach, Switzerland) was added to the plates and thereaction stopped after two to six minutes by adding H₂SO₄. Absorbancewas then read at 450 nm by a microplate reader (Biotek, USA;distributor: WITTEC A G, Littau, Switzerland). FIG. 1 shows that theparental hybridoma supernatants of various clones recognize the humanTL1A-his coated protein and not irrelevant his-tagged protein.

TNFRSF25 Blocking ELISA

The recombinant human TNFRSF25 receptor protein (TNFRSF25) was generatedas follows: the cDNA for human TNFRSF25 (clone name: IRCMp5012F0812D)was purchased from Source Biosystems (Nottingham, UK) and theextracellular portion (amino acids 25-199) of human TNFRSF25 (numberingaccording to the Uniprot Q93038 sequence) was amplified with flankingrestriction sites. The resulting PCR product encompassing an N-terminal8-His tag sequence was subsequently cloned into a modified version ofthe pcDNA3.1 vector from Invitrogen (Invitrogen AG, Basel, Switzerland)carrying a CMV promoter, a Bovine Growth Hormone poly-adenylation, andthe murine VJ2C leader peptide to drive the secretion of the recombinantprotein. For recombinant protein production, the recombinant vector wastransfected into suspension-adapted HEK 293 cells (ATCC number CRL 1573)using jetPEI™ transfection reagent (Polyplus-transfection S. A.,Strasbourg, France; Distributor: Brunschwig, Basel, Switzerland). Cellculture supernatant was collected after five days and purified using aProtein A affinity purification column (HiTrap Protein A sepharosecolumn; GE Healthcare Europe GmbH, Glattbrugg, Switzerland) operated onan AKTA FPLC system (GE Healthcare Europe GmbH, Glattbrugg,Switzerland).

In order to determine if the generated anti-TL1A antibodies can blockthe binding of TL1A to the TNFRSF25 receptor, a blocking ELISA wasdeveloped. Ninety-six well-microtiter plates (Costar, USA; distributorVWR A G, Nyon, Switzerland) were coated with 100 μl of recombinant humanTL1A-Fc (see Example 1) at 2 μg/ml in PBS or recombinant untagged TL1A(R&D Systems, Minneapolis, USA). Plates were incubated overnight at 4°C. and were then blocked with PBS 2% BSA at RT for one hour. Theblocking solution was removed and the hybridoma supernatants or purifiedantibodies were added to the plate. Five minutes later, 50 μl ofrecombinant human TNFRSF25-Fc-his (R&D Systems) at 4 μg/ml was added toeach well. Plates were incubated at RT for 60 minutes, then washed ninetimes with PBS 0.01% Tween-20 and mouse anti-poly histidine-HRP(Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) was added at a dilutionof 1:2000. Plates were incubated for 30 minutes at RT, washed 9 timeswith PBS 0.01% Tween-20 and the TMB substrate (Bio-rad Laboratories AG,Reinach, Switzerland) was added to the plates and the reaction stoppedafter 6 minutes by adding H₂SO₄. Absorbance was then read at 450 nm by amicroplate reader (Biotek, USA; distributor: WITTEC A G, Littau,Switzerland). FIG. 2 shows that the purified antibodies are able toblock the interaction between TL1A and TNFRSF25 in a dose dependentmanner.

Inhibition of TL1A-Induced IFN-γ Secretion by Primed CD4 T Cells

CD4 T cells primed by IL-12 and IL-18 cytokines polarize toward a TH1phenotype and secrete IFN-γ. TL1A has been shown to enhance IFN-γproduction by primed CD4 T cells. We therefore tested if the chimeric5G6 antibody could block this TL1A-dependent increase in IFN-γproduction.

To purify human CD4 T cells from peripheral blood mononuclear cells(PMBC), filters containing human leukocytes were collected from theBlood Collection Centre from La Chaux-de-Fonds, Switzerland (Centre deTransfusion Sanguine et Laboratoire de Sérologie, rue Sophie-Mairet 29,CH-2300). Cells were removed from the filters by back flushing with 60mL of PBS containing 10 U/mL of liquemin (Drossapharm AG, Lucern,Switzerland). PBMCs were then purified with 50 mL Blood-Sep-Filter Tubes(distributor: Brunschwig, Basel, Switzerland) following manufacturer'sinstructions. Cells were washed three times with phosphate bufferedsaline (PBS) and then used for CD4 purification using naïve CD4 T cellpurification kit from Miltenyi (Gladbach, Germany) according to themanufacturer's instructions.

The 5G6 antibody was tested to determine whether it could inhibit theeffect of naturally produced TL1A. IL-12 and IL-18 primed CD4 T cellswere incubated with monocytes that had been pre-activated by immunecomplexes (IC), and the production of IFN-γ was measured. Monocytes wereisolated from PBMCs (see above) using a monocyte isolation kit II fromMiltenyi (Gladbach, Germany) according to the manufacturer'sinstructions. Monocyte IC stimulation was performed as follows:chrompure human IgG (Jackson ImmunoResearch Europe Ltd, Newmarket, UK)was coated on a 12-well cell-culture plate (TPP, Trasadingen,Switzerland) at 50 μg/mL for 2 hrs at room temperature in PBS. The platewas then washed with PBS and incubated with mouse anti-human IgG(Jackson ImmunoResearch) for 1 hr at room temperature. The coated platewas washed once with PBS before plating of the purified monocytes.IC-stimulated monocytes were harvested after 48-72 hrs incubation at 37°C. in a 5% CO₂ incubator.

For flow cytometry analysis, IC-stimulated monocytes were stained withan anti-TL1A-PE antibody (GeneTex, distributed by Lucerna Chem. AG,Lucerna, Switzerland) or a rabbit isotype control (BD Pharmingen,Allschwil, Switzerland) at 10 μg/mL in a V-bottom 96-well microtiterplate (TPP, Trasadingen, Switzerland) at 4° C. for 30 min. The dilutionbuffer (FACS buffer) was PBS supplemented with 2% fetal calf serum (FCS,Amimed distributed by Bioconcept, Allschwil, Switzerland) and 10%Versene (Gibco Life Technologies). After incubation, 100 μL of FACSbuffer was added to each well and the plate was centrifuged at 300 g for3 min. Supernatant was discarded and samples were resuspended in 100 μLof a PE-anti rabbit secondary antibody solution at 0.2 μg/mL in FACSbuffer. Samples were incubated at 4° C. for 20 min. The plate was washedas described above and samples were resuspended in 300 μL of FACS bufferand immediately acquired on a FACSCyan flow cytometer (Beckman CoulterInternational S.A., Nyon, Switzerland). For soluble TL1A quantification,supernatant from IC-stimulated monocyte culture were harvested atdifferent time points (20, 48 and 72 hours) and sTL1A was quantifiedusing human TL1A development ELISA kit (Peprotech), according to themanufacturer's recommendations.

For the monocyte-T cell co-culture, IC stimulated monocytes (10⁴) andCD4 purified T cells (10⁵) were seeded in flat bottom-96 well plates(TPP) with IL-12 (Peprotech) at 8 ng/mL and IL-18 (MBL International,distributed by LabForce AG, Nunningen, Switzerland) at 200 ng/mL in aCO₂ incubator at 37° C. The supernatants of culture were harvested after72 hrs and IFN-γ was quantified as described above.

IC stimulated monocytes expressed TL1A on their membrane (mTL1A) (FIG.3A) but more substantially as a soluble factor (sTL1A) (FIG. 3B) andinduced a strong production of IFN-γ by co-cultured CD4 T cells (FIG.3C). The 5G6 antibody suppressed completely the production of IFN-γinduced by the IC-stimulated monocytes showing a potent blocking of bothmTL1A and sTL1A-mediated effect.

Example 4 Parental 5G6 Candidate Binds to Mouse, Rat, Cynomologus Monkeyand Human TL1A

The reactivity of the parental 5G6 antibody (produced as a chimericantibody with Human Fc) on the extracellular part of TL1A from diversespecies was tested by ELISA. The extracellular part of TL1A proteincorresponding to human (homo sapiens), rat (ratus norvegicus), mouse(mus musculus) and cynomologus monkey (macaca fascicularis) sequences(SEQ ID NOS: 38, 39, 40 and 41, respectively) was immobilized on highbinding 96-well plates (Costar, USA; distributor VWR AG, Nyon,Switzerland) overnight at a concentration of 2 μg/mL at 4° C. in PBS.Plates were blocked with 2% BSA albumin (BSA, Sigma-Aldrich Chemie,Buchs, Switzerland) at RT for one hour. The blocking solution wasremoved and a dose dilution of 5G6 antibody was applied to the plates.Plates were incubated at RT for 60 minutes, washed six times with PBS0.01% Tween-20 and the TMB substrate (Bio-rad Laboratories AG, Reinach,Switzerland) was added to the plates. The reaction was stopped after 6minutes by adding H₂SO₄ and the binding of 5G6 on the different TL1Aproteins was revealed using an HRP-labelled anti human IgG secondaryantibody (Sigma-Aldrich Chemie, Buchs, Switzerland), added at a dilutionof 1:1000. The plates were read for absorbance at 450 nm by a microplatereader (Biotek, USA; distributor: WITTEC A G, Littau, Switzerland). FIG.4 shows that 5G6 recognized, in a dose dependent manner, the TL1Aprotein from all tested species.

Example 5 Humanization of Mouse Monoclonal Antibody 5G6

Humanizing the anti-human TL1A mouse antibody 5G6 including selection ofhuman acceptor frameworks, back mutations, and mutations thatsubstantially retain and/or improve the binding properties of humanCDR-grafted acceptor frameworks is described herein.

Design of the Reshaped Variable Regions

Homology matching was used to choose human acceptor frameworks to graft5G6 CDRs. Databases e.g. a database of germline variable genes from theimmunoglobulin loci of human and mouse (the IMGT database, supra) or theVBASE2 (Rater I et al., (2005) Nucleic Acids Res. 33, Database issueD671-D674) or the Kabat database (Johnson G et al., (2000) Nucleic AcidsRes. 28: 214-218) or publications (e.g., Kabat E A et al., supra) may beused to identify the human subfamilies to which the murine heavy andlight chain V regions (SEQ ID NO: 1 and 2, respectively) belong anddetermine the best-fit human germline framework to use as the acceptormolecule. Selection of heavy and light chain variable sequences (VH andVL) within these subfamilies to be used as acceptor may be based uponsequence homology and/or a match of structure of the CDR1 and CDR2regions to help preserve the appropriate relative presentation of thesix CDRs after grafting.

For example, use of the IMGT database indicates good homology betweenthe 5G6 heavy chain variable domain framework and the members of thehuman heavy chain variable domain subfamily 1. Highest homologies andidentities of both CDRs and framework sequences were observed forgermline sequences: IGHV1-2*02 (SEQ ID NO: 3), IGHV1-2*04 (SEQ ID NO:4), IGHV1-2*05 (SEQ ID NO: 5), IGHV1-2*01 (SEQ ID NO: 6), andIGHV1-46*01 (SEQ ID NO: 7), all of which had sequence identity above 67%for the whole sequence up to CDR3. IGHV1-2*02 and IGHV1-2*04 showed69.4% sequence identity while IGHV1-2*01 and IGHV1-46*01 showed asequence identity of 68.4 and 67.3%, respectively.

Using the same approach, 5G6 light chain variable domain sequence showedgood homology to the members of the human light chain variable domainkappa subfamily 1. Highest homologies and identities of both CDRs andframework sequences were observed for germline sequences: IGKV1-33*01(SEQ ID NO: 8) and IGKV1D-33*01 (SEQ ID NO: 9) exhibited the highestidentity (both having 80.0% identity), closely followed by another groupconsisting of IGKV1D-12*02 (SEQ ID NO: 10), IGKV1D-12*01 (SEQ ID NO:11), and IGKV1-12*02 (SEQ ID NO: 12) all exhibiting the same degree ofsequence identity (75.8%).

As starting point to the humanization process, human IGHV1-2*01 (SEQ IDNO: 3), and IGKV1-33*01 (SEQ ID NO: 8) variable domains were selected asacceptors to the 5G6 CDRs. A first humanized antibody of human gamma oneisotype was prepared (see below). The antibody encompassed a human-mousehybrid heavy chain variable domain and a human-mouse hybrid light chainvariable domain. The hybrid heavy chain variable domain was based on thehuman heavy chain variable domain IGHV1-2*01 wherein germline CDR1 and 2where respectively replaced for 5G6 heavy chain CDR1 and 2. Bestmatching JH segment sequence to the human acceptor framework wasidentified from the IMGT searches mentioned above. The resultinghuman-mouse hybrid heavy chain variable sequence having human IGHV1-2*01framework regions, 5G6 mouse CDRs, and best matching JH to humanacceptor is refereed herein as heavy chain variable domain VH1 with SEQID NO: 13. Similarly, the human-mouse hybrid light chain variable domainused for this first humanized antibody candidate had human IGKV1-33*01framework regions, 5G6 mouse CDRs, and best matching JK to humanacceptor, and is refereed herein as light chain variable domain VL1 withSEQ ID NO: 14. The first humanized antibody encompassing VH1 and VL1 isabbreviated herein VH1/VL1 antibody.

Production of the First Humanized Antibody Prototype

Coding DNA sequences (cDNAs) for VH1 and VL1 were synthesized in a scFvformat by GENEART AG (Regensburg, Germany) thereby allowing for a singlecDNA sequence to encompass both variable domains (SEQ ID NO: 15).Individual variable domain cDNAs were retrieved from this scFv constructby PCR, and further assembled upstream of their respective constantdomain cDNA sequence(s) using PCR assembly techniques. Finally, thecomplete heavy and light chain cDNAs were ligated in independent vectorsthat are based on a modified pcDNA3.1 vector (Invitrogen, Calif., USA)carrying the CMV promoter and a Bovine Growth Hormone poly-adenylationsignal. The light chain specific vector allowed expression of humankappa isotype light chains by ligation of the light chain variabledomain cDNA of interest in front of the kappa light chain constantdomain cDNA using BamHI and BsiWI restriction enzyme sites; while theheavy chain specific vector was engineered to allow ligation of theheavy chain variable domain cDNA of interest in front of the cDNAsequence encoding the human IGHG1 CH1, IGHG1 hinge region, IGHG1 CH2,and IGHG1 CH3 constant domains using BamHI and SalI restriction enzymesites. In both heavy and light chain expression vectors, secretion wasdriven by the mouse VJ2C leader peptide containing the BamHI site. TheBsiWI restriction enzyme site is located in the kappa constant domain;whereas the SalI restriction enzyme site is found in the IGHG1 CH1domain.

The VH1/VL1 antibody (having heavy chain SEQ ID NO: 16 and light chainSEQ ID NO: 17) was transiently produced by co-transfecting equalquantities of heavy and light chains vectors into suspension-adaptedHEK293-EBNA1 cells (ATCC® catalogue number: CRL-10852) usingpolyethylenimine (PEI, Sigma, Buchs, Switzerland). Typically, 100 ml ofcells in suspension at a density of 0.8-1.2 million cells per ml istransfected with a DNA-PEI mixture containing 50 μg of expression vectorencoding the heavy chain and 50 μg of expression vector encoding thelight chain. When recombinant expression vectors encoding antibody genesare introduced into the host cells, antibodies are produced by furtherculturing the cells for a period of 4 to 5 days to allow for secretioninto the culture medium (EX-CELL 293, HEK293-serum-free medium; Sigma,Buchs, Switzerland), supplemented with 0.1% pluronic acid, 4 mMglutamine, and 0.25 μg/ml geneticin).

The VH1/VL1 antibody was purified from cell-free supernatant usingrecombinant protein-A streamline media (GE Healthcare Europe GmbH,Glattbrugg, Switzerland), and buffered exchanged into phosphate buffersaline prior to assays.

Kinetic Binding Affinity Constants by Surface Plasmon Resonance (SPR)

Kinetic binding affinity constants (KD) were measured on protein-Acaptured antibody using recombinant histidine tagged TL1A as analyte.Measurements were conducted on a BIAcore 2000 (GE Healthcare—BIAcore, GEHealthcare Europe GmbH, Glattbrugg, Switzerland) at room temperature,and analyzed with the BiaEvaluation software (BIAcore; v4.1, GEHealthcare Europe GmbH).

A CM5 research grade sensor chip (GE Healthcare Europe GmbH; ref.BR-1000-14) was activated by injecting 35 ml of a 1:1N-hydroxysulfosuccinimide(NHS)/1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC)solution (v/v; 5 μl/min flow-rate; on flow paths 1 and 2). Protein-A(ref. P7837; Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) was dilutedto a final concentration of 50 μg/ml in acetate buffer pH 4.5 (GEHealthcare Europe GmbH, BR-1003-50; one pH unit below pI) andsubsequently immobilized on the previously activated CMS sensor chip byinjecting 35 μl on both flow path 1 and 2 (5 μl/min); this correspondedto approximately 1500 response units (RUs). The protein-A-CMS sensorchip was then deactivated by injecting 35 μl of ethanolamine solution (5μl/min). Finally, two injections of 10 μl of glycine solution (GEHealthcare Europe GmbH, ref BR-1003-54; 10 mM; pH 1.5) were performed torelease non-crosslinked protein-A molecules.

For affinity measurements, the chimeric and humanized antibody stored in1× PBS buffer were diluted in HBS-EP buffer (GE Healthcare Europe GmbH,ref BR-1001-88; 0.01 M HEPES, 0.15 M NaCl, EDTA 3 mM, 0.005% SurfactantP20, pH 7.4) and subsequently injected on the flow-path 2 of theprotein-A CMS chip (30 μl/min) to reach about 180 RUs. Following thiscapture step, the recombinant histidine tagged human TL1A was injectedat different concentrations (1.25 to 125 nM) on the flow-path 1 and 2(flow-path 1 being used as reference) at a 30 μl min flow rate. Aftereach binding event, surface was regenerated with glycine buffer pH 1.5injected for 20 sec (30 μl/min).

Measurements (sensorgram: fc2-fc1) were best fitted with a 1:1 analytemodel without mass transfer. To account for the experimental variationsin protein-A captured antibody at the beginning of each measurement, theRmax value was set to local in all fits. Dissociation times were of atleast 300-600 seconds. Measurements were performed in duplicate andincluded zero-concentration samples for referencing. The Chi2 valuerepresents the sum of squared differences between the experimental dataand reference data at each point; while the plots of residuals indicatethe difference between the experimental and reference data for eachpoint in the fit. Both Chi2 and residual values were used to evaluatethe quality of a fit between the experimental data and individualbinding models.

Back Mutations of Grafted Human Frameworks

Since straight grafting of CDRs from 5G6 mouse antibody led to acandidate having no binding to human TL1A (Table 6), mutagenesis whereinhuman residues are substituted for mouse residues was initiated. Thisprocess is called back-mutation and is the most unpredictable procedurein the humanization of monoclonal antibodies. It necessitates theidentification and the selection of critical framework residues from themouse antibody that need to be retained in order to preserve affinitywhile at the same time minimizing potential immunogenicity in thehumanized antibody.

To identify residues that may impact the most CDR conformation and/orinter-variable domain packing, a 3D model for the VH1-VL1 pair ofvariable domains was calculated using the structure homology-modellingserver SWISS-MODEL (Arnold K et al., (2006) Bioinformatics, 22(2):195-201) set in automated mode. Model analysis allowed the selection ofa subset of positions based on their putative influence on CDR regionsand/or heavy chain-light chain variable domain packing. This subset ofpositions consisted of variable heavy chain positions: 37, 48, 50, 67,69, 71 and 75 as well as variable light chain positions: 5 and 34 (Kabatnumbering).

Further humanized candidates having back mutations at the selectedpositions mentioned above were prepared using gene synthesis andstandard mutagenesis methods. A single cDNA sequence encompassing bothVH2 and VL2 variable domains (SEQ ID NO: 18) was synthesised and used asa starting point for further mutagenesis. Antibody expression andpurification followed the methods described above. Humanized antibodycandidates were assayed for their binding affinity by SPR as previouslydescribed.

Binding properties (KD) of selected humanized antibodies based on thesesingle or combination of substitutions are shown in Table 6. Amongsthumanized variants, VH3/VL1 antibody had the highest affinity for TL1Aantigen, exhibiting a lower KD than the 5G6 chimeric antibody.

Thermostability of Selected Humanized Anti-TL1A Antibodies byDifferential Scanning Calorimetry

The thermal stabilities of the humanized antibodies were measured usingdifferential scanning calorimetry (DSC). Monoclonal antibodies meltingprofiles are characteristic of their isotypes (Garber E & Demarest S J,(2007) Biochem. Biophys. Res. Commun. 355: 751-7), however the mid-pointmelting temperature of the FAB fragment can be easily identified even inthe context of a full-length IgG. Such mid-point melting of FAB portionwas used to monitor monoclonal stability of the humanized candidates.

Calorimetric measurements were carried out on a VP-DSC differentialscanning microcalorimeter (GE Healthcare Europe GmbH). The cell volumewas 0.128 ml, the heating rate was 200° C./h, and the excess pressurewas kept at 65 p.s.i. All antibodies were used at a concentration of 1mg/ml in PBS (pH 7.4). The molar heat capacity of antibody was estimatedby comparison with duplicate samples containing identical buffer fromwhich the antibody had been omitted. The partial molar heat capacitiesand melting curves were analyzed using standard procedures. Thermogramswere baseline corrected and concentration normalized before beingfurther analyzed using a Non-Two State model in the software Originv7.0.

Humanized variant VH5/VL1 FAB fragment displayed a single transition at83.8° C. with a shape and amplitude consistent with a cooperativeunfolding which is generally observed for a compactly folded FABfragments indicating that the engineering process was successful atretaining FAB stability. Overall the humanized variant showed a goodthermal stability.

TABLE 6 humanized anti human TL1A antibodies Antibody SEQ variant IDBack-mutations K_(D) (IGHG1) NOs VH/VL (pM) Chimera 19, 20 N.A./N.A. 728VH1/VL1 16, 17 N.A./N.A. no binding VH2/VL2 21, 25 VH: V37A-M48I-W50E-857 V67A-M69L-R71V-I75S VL: N34S-T5N VH3/VL1 22, 17 VH:V37A-M48I-W50E-V67A-R71V 249 VH4/VL1 23, 17 VH: V37A-M48I-W50E-R71V 681VH5/VL1 24, 17 VH: V37A-W50E-R71V 259

Example 6 5G6 Humanized Candidates can Block TL1A-Induced IFN-γSecretion by Primed CD4 T Cells

Humanized candidates of the 5G6 antibody were tested to determinewhether they could inhibit the TL1A-dependent increase in IFN-₁production.

Human CD4 T cells were purified from peripheral blood mononuclear cells(PMBC) as described in Example 3 above. All cell cultures were performedin Roswell Park Memorial Institute medium (RPMI-1640, PAA Laboratories,Pasching, Austria) supplemented with 10% heat inactivated foetal calfserum (FCS, Amimed distributed by Bioconcept, Allschwil, Switzerland),non essential amino acids (PAA, distributed by Chemie Brunschwig AG,Basel, Switzerland), ultraglutamine (Lonza, Basel, Switzerland), sodiumpyruvate (PAA) and penicillin/streptomycin mix (Gibco Lifetechnologies). CD4 purified T cells (10⁵ cells/well) were incubated withIL-12 (Peprotech, Hamburg Germany) at 8 ng/mL, IL-18 (MBL International,distributed by LabForce AG, Nunningen, Switzerland) at 200 ng/mL(priming factors) and human soluble TL1A with an N-terminal his-tag(encoded by SEQ ID NO: 118) for 72 hrs in presence of the blocking 5G6humanized antibody candidates (VH3/VL1—SEQ ID NOS: 22 and 17,VH4/VL1—SEQ ID NOS: 23 and 17, VH5/VL1—SEQ ID NOS: 24 and 17 andVH2/VL2—SEQ ID NO: 21 and 25), added at the concentrations 100, 10, 1and 0.1 μg/mL (see tables in FIG. 5), at the same time. The isotypecontrol was added at 100 m/mL in a flat bottom 96-well cell cultureplate (TPP AG, Trasadingen, Switzerland). The supernatants wereharvested after 72 hrs and IFN-γ was quantified by ELISA using OptEIAkit (BD Pharmingen, Allschwil, Switzerland) according to themanufacturer's instructions. FIG. 5 shows that humanized anti-TL1Aantibodies were able to inhibit substantially the production of IFN-γ.

Example 7 5G6 Humanized Antibody is Effective in a Murine Model ofAllergic Asthma

Asthma does not spontaneously develop in mice therefore to investigatethis disease in mice an asthmatic-like reaction needs to be induced inthe airways. A variety of acute allergen challenge models have beendeveloped and in this example BALB/c mice were used as they develop agood T helper cell 2 (Th2)-biased immunological response (Boyce J A &Austin K F (2005) J Exp Med, 201: 1869-73). Ovalbumin, derived fromchicken egg is an allergen that induces a robust, allergic pulmonaryinflammation in mice and therefore is frequently used in murine modelsof allergic asthma (Kumar R K et al., (2008) Curr Drug Targets, 9:485-94).

In this example, the following immunization protocol was used to induceallergic asthma: BALB/c mice were sensitized by i.p. injection of 100 μgof ovalbumin (Albumin from chicken egg white, Grade V, Sigma Aldrich,Switzerland) adsorbed on 1 mg of a suspension of aluminium hydroxide andmagnesium hydroxide (Imject Alum, Thermo Scientific, Switzerland) on day0 and day 14. On day 28, 30 and 33, mice were treated i.p. with 50 mg/kgof humanized 5G6 antibody (VH5/VL1; format IgG4 hinge stabilised; SEQ IDNO: 124 and 17) or an equivalent amount of the control human IgG. As apositive control, dexamethasone (Sigma, Switzerland) was used at 5mg/kg. Four hours following treatment, mice were anesthetized with 30mg/kg of xylazine and 150 mg/kg of Ketamin (Xylazol and Ketasol fromGraeub Veterinary products, Switzerland) and injected intranasally with10 μg of ovalbumin. Three days later, mice were sacrificed and aftercannulation of their trachea, bronchoalveolar lavage (BAL) was performedby injecting 2 ml of cold PBS into the lung. Cells in BAL fluids werecounted and eosinophils detected by flow cytometry using CD11c andSiglec F cell surface markers.

The results are shown in FIG. 6 where it can be observed that treatmentwith the humanized antibody 5G6 resulted in approximately a 4-foldreduction in the number of eosinophils in BAL fluid of asthmatic mice.

Example 8 5G6 Humanized Antibody is Effective in a Treating DSS-InducedAcute Colitis in Mice

Many different animal models of inflammatory bowel disease (IBD) havebeen developed and these are valuable tools for investigating theinvolvement of various factors into the pathogenesis of IBD and toevaluate therapeutic options. The dextran sulphate sodium (DSS) inducedcolitis model is a widely used model of inflammatory bowel diseasebecause of its simplicity and it has many similarities to human IBD,particularly ulcerative colitis (Perk M & Cerar A (2012) J BiomedBiotechnol, 2012; 718617; Wirtz S et al., (2007) Nat. Protoc, 2: 541-6).

To evaluate the potential effect of humanized 5G6 antibody (VH5/VL1;format IgG4 hinge stabilised; SEQ ID NO: SEQ ID NO: 124 and 17) in IBD,a condition of acute colitis was induced in C57B1/6 mice byadministering 2% of DSS (MW 36-50 kDa; MP Biomedicals) in the drinkingwater of the test group for 5 days. The control group were givenuntreated tap water. Following DSS exposure, tap water was given to thetest group of mice for 7 days. Mice were treated i.p 3×/week with 50mg/kg of humanized 5G6 antibody or an equal amount of isotype control.As a positive control, cyclosporine (Sandimmune, Novartis Pharma,Switzerland) was used at 5 mg/kg. Mice were monitored daily for weightloss and stool consistency. At day 12, all mice were sacrificed andtheir entire colon lengths measured. As shown in FIG. 7, it can beobserved that treatment of mice with humanized antibody 5G6 resulted inreduction of colon shortening induced by DSS.

Example 9 5G6 Humanized Antibody is Effective in a Treating TNBS Colitisin Rats

Intestinal inflammation in rats can be induced by intrarectaladministration of trinitrobenzenesulfonic acid (TNBS). The resultinglocalised ulceration and inflammation is believed to involve a T-cellmediated response against hapten-modified autologous proteins or luminalantigens (Wirtz S et al., supra). Symptoms include diarrhoea, occultblood and weight loss.

To evaluate the potential effect of a humanized 5G6 antibody (VH5/VL1;format IgG4 hinge stabilised; SEQ ID NO: SEQ ID NO: 124 and 17) in IBD,a condition of colitis was induced in Sprague-Dawley rats by intrarectaladministration of TNBS Solution (50% TNBS: 50% 200 proof ethanol; 16mg/ml TNBS (Sigma, Cat#92822) at 64 mg/kg (4 ml/kg) into the colon ofanaesthetised rats in the treatment groups. The control group receivedno TNBS Solution. Two hours after TNBS administration, rats were treatedi.p with a single dose of humanized 5G6 antibody (50 mg/kg) or an equalamount of isotype control. As a positive control, prednisolone (Sigma)was administered orally at a dose of 10 mg/kg two hours after TNBSadministration and daily for the following 5 days. Rats were sacrificedon day 7 and disease severity assessed as a colonic score using thefollowing scoring system:

-   1) Adhesions: none=0, minimal=1, involving several bowel loops=2-   2) Strictures: none=0, mild=2, severe, proximal dilatation=3-   3) Ulcers: none=0, linear ulceration<1 cm=1, two linear ulcers<1    cm=2, more sites of ulceration or one large ulcer=3-   4) Wall thickness: less than 1 mm=0, 1-3 mm=1, >3 mm=2

As shown in FIG. 8, it can be observed that treatment of rats withhumanized antibody 5G6 resulted in reduction of disease parametersinduced by TNBS.

Example 10

Binding of 5G6 Humanized Antibody to hTL1A is Blocked by Both hDcR3-Fcand hDR3-Fc

As discussed above TL1A is a ligand for TNFRSF25/DR3 and the decoyreceptor DcR3. DR3 is a death domain-containing receptor that isupregulated during T cell activation and the interaction of TL1A withDR3 can promote T cell expansion during an immune response (Migone T Set al., supra). The secreted decoy receptor (DcR3), a soluble protein ofthe tumor necrosis factor receptor (TNFR) superfamily, blocks the actionof TL1A. (Kim S & Zhang L, supra).

To evaluate whether the 5G6 humanized antibody could interfere with theinteraction of hTL1A to the receptor DR3 and/or to the decoy receptorDcR3, a his-tagged human TL1A was coated at 2 μg/ml on an ELISA plateand incubated with 20 μg/ml of 5G6 humanized antibody (VH5/VL1; formatIgG4 hinge stabilised; SEQ ID NO: SEQ ID NO: 124 and 17) in the presenceof 10 μg/ml of Fc fusions of the ectodomains of either human DcR3, DR3(both R&D systems) or an irrelevant receptor (Ctrl-Fc) followed bydetection with peroxidase-conjugated anti-human IgG (Fab specific). Ascan be observed in FIG. 9, binding of the 5G6 humanized antibody tohTL1A is blocked by both hDcR3-Fc and hDR3-Fc. This confirms that 5G6humanized antibody binding to hTL1A inhibits the interaction of hTL1Awith both DR3 and DcR3.

1. An antibody or fragment thereof that binds to TL1A comprising a heavychain CDR1 comprising the amino acid sequence of SEQ ID NO: 51, a heavychain CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and aheavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 53;and/or comprising a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 54, a light chain CDR2 comprising the amino acid sequenceof SEQ ID NO: 55, and a light chain CDR3 comprising the amino acidsequence of SEQ ID NO:
 56. 2. The antibody or fragment thereof of claim1, wherein the antibody or fragment thereof is a murine antibody,chimeric antibody or a humanized antibody.
 3. The antibody or fragmentthereof of claim 1, wherein the antibody or fragment thereof is ahumanized antibody.
 4. The antibody or fragment thereof of claim 1,wherein the antibody or fragment thereof comprises a heavy chainvariable region sequence comprising the amino acid sequence of SEQ IDNO:
 1. 5. The antibody or fragment thereof of claim 1, wherein theantibody or fragment thereof comprises a non-CDR region of a heavy chainvariable region sequence which is at least 80% identical to the non-CDRregion of the heavy chain variable region sequence of SEQ ID NO:
 1. 6.The antibody or fragment thereof of claim 1, wherein the antibody orfragment thereof comprises a heavy chain variable region sequencecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOS: 26, 27, 28 and
 29. 7. The antibody or fragment thereof ofclaim 1, wherein the antibody or fragment thereof comprises a non-CDRregion of a heavy chain variable region sequence which is at least 80%identical to the non-CDR region of the heavy chain variable regionsequence selected from the group consisting of SEQ ID NOS: 26, 27, 28and
 29. 8. The antibody or fragment thereof of claim 1, wherein theantibody or fragment thereof comprises a heavy chain variable regionsequence comprising the amino acid sequence of SEQ ID NO:
 29. 9. Theantibody or fragment thereof of claim 1, wherein the antibody orfragment thereof comprises a non-CDR region of a heavy chain variableregion sequence which is at least 80% identical to the non-CDR region ofthe heavy chain variable region sequence of SEQ ID NO:
 29. 10. Theantibody or fragment thereof of claim 1, wherein the antibody orfragment thereof comprises a heavy chain sequence comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS: 21, 22,23 and
 24. 11. The antibody or fragment thereof of claim 1, wherein theantibody or fragment thereof comprises a heavy chain sequence comprisinga non-CDR region which is at least 80% identical to the non-CDR regionof the heavy chain variable region sequence of a heavy chain sequenceselected from the group consisting of SEQ ID NOS: 21, 22, 23 and
 24. 12.The antibody or fragment thereof of claim 1, wherein the antibody orfragment thereof comprises a heavy chain variable framework region thatis the product of or derived from a human gene selected from the groupconsisting of IGHV1-2*02 (SEQ ID NO: 3), IGHV1-2*04 (SEQ ID NO: 4),IGHV1-2*05 (SEQ ID NO: 5), IGHV1-2*01 (SEQ ID NO: 6), and IGHV1-46*01(SEQ ID NO: 7).
 13. The antibody or fragment thereof of claim 3, whereinthe antibody or fragment thereof comprises a heavy chain variableframework region that is the product of or derived from human geneIGHV1-2*01 (SEQ ID NO: 3) and wherein the heavy chain variable frameworkregion comprises at least one amino acid modification from thecorresponding heavy chain variable framework region of a correspondingmurine antibody.
 14. The antibody or fragment thereof of claim 3,wherein the antibody or fragment thereof comprises a heavy chainsequence comprising the amino acid sequence of SEQ ID NO: 16 and whereinthe heavy chain variable framework region comprises at least one aminoacid modification from the corresponding heavy chain variable frameworkregion of a corresponding murine antibody.
 15. The antibody or fragmentthereof of claim 13 or 14, wherein the amino acid modification comprisesan amino acid substitution at amino acid position selected from thegroup consisting of 37, 48, 50, 67, 69, 71 and 75, wherein the aminoacid position of each group member is indicated according to the Kabatnumbering.
 16. The antibody or fragment thereof of claim 13 or 14,wherein the amino acid modification comprises an amino acid substitutionselected from the group consisting of 37A, 48I, 50E, 67A, 69L, 71V and75S, wherein the amino acid position of each group member is indicatedaccording to the Kabat numbering.
 17. The antibody or fragment thereofof claim 1, wherein the antibody or fragment thereof comprises a lightchain variable region sequence comprising the amino acid sequence of SEQID NO:
 2. 18. The antibody or fragment thereof of claim 1, wherein theantibody or fragment thereof comprises a non-CDR region of a light chainvariable region sequence which is at least 80% identical to the non-CDRregion of the light chain variable region sequence of SEQ ID NO:
 8. 19.The antibody or fragment thereof of claim 1, wherein the antibody orfragment thereof comprises a light chain variable region sequencecomprising the amino acid sequence selected from the group consisting ofSEQ ID NOS: 14 and
 30. 20. The antibody or fragment thereof of claim 1,wherein the antibody or fragment thereof comprises a non-CDR region of alight chain variable region sequence which is at least 80% identical tothe non-CDR region of the light chain variable region sequence selectedfrom the group consisting of SEQ ID NO: 14 and
 30. 21. The antibody orfragment thereof of claim 1, wherein the antibody or fragment thereofcomprises a light chain sequence comprising the amino acid sequenceselected from the group consisting of SEQ ID NO: 17 and
 25. 22. Theantibody or fragment thereof of claim 1, wherein the light chainsequence comprises a non-CDR region which is at least 80% identical tothe non-CDR region of the light chain variable region sequence of thelight chain sequence selected from the group consisting of SEQ ID NO: 17and
 25. 23. The antibody or fragment thereof of claim 1, wherein theantibody or fragment thereof comprises a light chain variable frameworkregion that is the product of or derived from a human gene selected fromthe group consisting of IGKV1-33*01 (SEQ ID NO: 8), IGKV1D-33*01 (SEQ IDNO: 9), IGKV1D-12*02 (SEQ ID NO: 10), IGKV1D-12*01 (SEQ ID NO: 11) andIGKV1-12*02 (SEQ ID NO: 12).
 24. The antibody or fragment thereof ofclaim 3, wherein the antibody or fragment thereof comprises a lightchain variable framework region that is the product of or derived fromhuman gene IGKV1-33*01 (SEQ ID NO: 8) and wherein the light chainvariable framework region comprises at least one amino acid modificationfrom the corresponding framework region of the light chain variableregion of a corresponding murine antibody.
 25. The antibody or fragmentthereof of claim 3, wherein the antibody or fragment thereof comprises alight chain sequence comprising the amino acid sequence of SEQ ID NO: 17and wherein the light chain variable framework region comprises at leastone amino acid modification from the corresponding light chain variableframework region of the corresponding murine antibody.
 26. The antibodyor fragment thereof of claim 24 or 25, wherein the amino acidmodification comprises an amino acid substitution at amino acid positionselected from the group consisting of 5 and 34, wherein the amino acidposition of each group member is indicated according to the Kabatnumbering.
 27. The antibody or fragment thereof of claim 24 or 25,wherein the amino acid modification comprises an amino acid substitutionselected from the group consisting of 5N and 34S, wherein the amino acidposition of each group member is indicated according to the Kabatnumbering.
 28. The antibody or fragment thereof of claim 1, wherein theantibody or fragment thereof comprises: (a) a heavy chain sequencecomprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 24;and (b) a light chain sequence comprising the amino acid sequence of SEQID NO:
 17. 29. The antibody or fragment thereof of claim 1, wherein theantibody or fragment thereof comprises: (a) a heavy chain variableregion sequence comprising the amino acid sequence of SEQ ID NO: 27 orSEQ ID NO: 29; and (b) a light chain variable region sequence comprisingthe amino acid sequence of SEQ ID NO:
 14. 30. The antibody or fragmentthereof of claim 1, wherein at least one of the heavy chain CDRs and/orat least one of the light chain CDRs comprises at least one amino acidmodification.
 31. The antibody or fragment thereof of claim 1, furthercomprising heavy and/or light constant regions.
 32. The antibody orfragment thereof of claim 31, wherein the human heavy constant region isselected from the group of human immunoglobulins consisting of IGHG1,non fucosylated IGHG1 and IGHG4.
 33. The antibody or fragment thereof ofclaim 1, wherein the antibody has a non fucosylated IGHG1 Fc region. 34.The antibody or fragment thereof of claim 1, wherein the antibodycomprises an isotypic variant comprising the CH1 from human IgG4(IGHG4), the hinge from human IgG4 (IGHG4), having S228P substitutionand the CH2 and CH3 from human IgG4 (IGHG4).
 35. The antibody orfragment thereof of claim 1, wherein the antibody or fragment thereofbinds to human TL1A.
 36. The antibody or fragment thereof of claim 1,wherein the antibody or fragment thereof binds to human TL1A and iscross reactive with murine, rat and cynomologus TL1A.
 37. The antibodyor fragment thereof of claim 1, wherein the antibody or fragment thereofbinds to hTL1A and inhibits the interaction of hTL1A with both DR3 andDcR3.
 38. The antibody or fragment thereof of claim 1, wherein theantibody or fragment thereof is an antagonist antibody.
 39. The antibodyor fragment thereof of claim 1, wherein the antibody or fragment thereofis a neutralizing antibody.
 40. The antibody or fragment thereof ofclaim 1, wherein the antibody is a monovalent antibody.
 41. The antibodyor fragment thereof of claim 1, wherein the antibody is a full lengthantibody.
 42. The antibody or fragment thereof of claim 1, wherein theantibody is an antibody fragment selected from the group consisting ofFab, Fab′, Fab′-SH, Fd, Fv, dAb, F(ab′)2, scFv, bispecific single chainFv dimers, diabodies, triabodies and scFv genetically fused to the sameor a different antibody.
 43. The antibody or fragment thereof of claim1, wherein the antibody comprises a variant Fc region which comprises atleast one amino acid modification relative to the Fc region of theparent antibody, wherein the antibody comprising the variant Fc regionexhibits altered effector function compared to the parent antibody. 44.The antibody or fragment thereof of claim 1, wherein the antibody orfragment thereof binds to human TL1A with an affinity (K_(D)) of 700 pMor less.
 45. The antibody or fragment thereof of claim 1 or 3, whereinthe antibody or fragment thereof retains at least 85% of the TL1Abinding affinity (K_(D)) of the corresponding chimeric antibody.
 46. Theantibody or fragment thereof of claim 3, wherein the antibody orfragment thereof has equivalent or higher TL1A binding affinity (K_(D))when compared to the corresponding chimeric antibody.
 47. The antibodyor fragment thereof of claim 3, wherein the antibody or fragment thereofbinds human TL1A with three-fold higher affinity than the correspondingchimeric antibody
 48. The antibody or fragment thereof of claim 1,wherein the antibody has a Fab fragment thermostability temperaturegreater than 80° C.
 49. An antibody or fragment thereof that binds tohuman TL1A and which binds to the same epitope as the antibody ofclaim
 1. 50. An epitope on soluble human TL1A which is bound by theantibody of claim
 1. 51. An isolated nucleic acid encoding the antibodyor fragment thereof of claim
 1. 52. The isolated nucleic acid of claim51 comprising DNA encoding the heavy chain variable region comprisingthe nucleic acid sequence of SEQ ID NO: 33 or 35; and/or DNA encodingthe light chain variable region comprising the nucleic acid sequence ofSEQ ID NO:
 36. 53. A vector comprising the isolated nucleic acid ofclaim
 52. 54. A host cell comprising the isolated nucleic acid of claim51 or the vector of claim
 53. 55. A method of producing an antibody orfragment thereof that binds to human TL1A comprising culturing the hostcell of claim 54 so that the nucleic acid is expressed and the antibodyproduced.
 56. An antibody or fragment thereof that binds to human. TL1Aencoded by the isolated nucleic acid of claim
 51. 57. A compositioncomprising the antibody or fragment thereof of claim 1 and apharmaceutically acceptable carrier.
 58. An immunoconjugate comprisingthe antibody or fragment thereof of claim 1 linked to a therapeuticagent.
 59. A composition comprising the immunoconjugate of claim 58 anda pharmaceutically acceptable carrier.
 60. The composition of claim 57further comprising a second pharmaceutically active agent.
 61. A methodfor treating a TL1A mediated disorder in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of the antibody or fragment thereof of claim
 1. 62. The method ofclaim 61, wherein the TL1A mediated disorder is selected from the groupconsisting of inflammatory bowel disease (IBD) including ulcerativecolitis and Crohn's disease, rheumatoid arthritis, MS, type 1 and type 2diabetes, psoriasis, psoriatic arthritis, ankylosing spondylitis, atopicdermatitis; allergic reactions or conditions, including for example,asthma and allergic lung inflammation; cancers, atherosclerosis,infections, neurodegenerative diseases, graft rejection, graft versushost diseases (GVHD) and cardiovascular disorders/diseases. transplantrejection, central nervous system injury, psoriasis, leukaemia orlymphoma (e.g., chronic lymphocytic leukaemia (CLL)), atherosclerosis,and lung and colon carcinomas, chronic obstructive pulmonary disease(COPD), optic neuritis, age related macular degeneration, systemic lupuserythematosus (SLE), sjogen's syndrome, scleroderma, systemic sclerosis,chronic Kidney disease, liver fibrosis, tuberculosis, idiopathicpulmonary fibrosis, tuberculosis induced lung fibrosis, retroperitonealFibrosis, pulmonary fibrosis, cystic fibrosis, endomyocardial fibrosis,atrial fibrosis, mediastinal fibrosis, myelofibrosis (bone marrow),retroperitoneal fibrosis, progressive massive fibrosis, npephrogenicsystemic fibrosis, and arthrofibrosis.
 63. The method of claim 61,wherein the antibody has a non fucosylated IGHG1 Fc region and exhibitsenhanced cytotoxicity as compared to the antibody having human heavychain constant region IGHG1.
 64. The antibody or fragment thereof ofclaim 1, for use as a medicament.
 65. The antibody or fragment thereofof claim 1, for use in a method for treating a TL1A mediated disorder.66. An article of manufacture comprising the antibody or fragmentthereof of claim 1, for the treatment of a TL1A mediated disorder.
 67. Akit comprising the antibody or fragment thereof of claim 1, for thetreatment of a TL1A mediated disorder.